VANILIN CAS N°: 121-33-5 - inchem.orginchem.org/documents/sids/sids/121335.pdf · oecd sids vanillin bli i 2 sids initial assessment profile cas no. 121-33-5 chemical name vanillin

OECD SIDS VANILIN

UNEP PUBLICATIONS

FOREWORD INTRODUCTION

VANILINCAS N°: 121-33-5

OECD SIDS VANILLIN

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SIDS INITIAL ASSESSMENT PROFILE

CAS No. 121-33-5

CHEMICAL NAME Vanillin

STRUCTURALFORMULA

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CONCLUSIONS

Only minor quantities of vanillin are released from the vanillin production sites. Some occupationalexposure to vanillin by inhalation of dust has been identified. The consumers will be directly exposedto vanillin as most of the produced material is ingested as flavour additive to food and beverages.

Environment:

Vanillin occurs widely in plants in the nature, usually as a glycoside bound to sugar or as a percusorto vanillin bound to the large lignin molecule abundant in wood. Free vanillin in the environment willbe distributed to the aqueous compartment, and there is no tendency to bioaccumulation. Theemission of vanillin from the vanillin production and from consumer products to the environment, isnot considered to represent any biohazard.

Human Health:

During the present reviewing of the available toxicity data for vanillin, no particular risk has beenidentified which should give reason to concern or additional toxicity testing in animals.

The use of vanillin as a food additive is approved by authorities world wide, and FDA has grantedGRAS status to its use. This is in agreement with the experience with vanillin in consumer productsduring many years without any confirmed report of adverse events.

RECOMMENDATIONSIt is recommended to make efforts to obtain more data on the occupational exposure throughmonitoring of inhalation of vanillin dust in contaminated working areas. Such monitoring datashould also include particles larger than those normally considered respirable.

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SIDS SUMMARYDATE: 20.08.96

CAS NO: 121-33-5Infor-mationavailable

OECDStudy

GLP OtherStudy

Esti-mationMethods

Accept-able

SIDSTestingRequired

STUDY Y/N Y/N Y/N Y/N Y/N Y/N Y/N

PHYSICAL-CHEMICAL

2.12.22.32.42.52.62.12

Melting PointBoiling PointDensityVapour PressurePartition CoefficientWater Solubility - pH and pKa valuesOxidation: Reduction Potential

YYYYYY

n.a.

NNNNNN

N/?N/?N/?

?N/?N/?

YYYYYY

NNNN

Y/NY/N

YYYYYY

NNNNNN

OTHER P/C STUDIES RECEIVED Flash point, Autoflammability, Flammability, Explosiveproperties, Oxidizing properties, Additional data

ENVIRONMENTAL FATE and PATHWAY

3.1.13.1.23.23.33.5

PhotodegradationStability in waterMonitoring dataTransport and DistributionBiodegradation

YYYYY

NYNYN

NY

n.a.n.a.N/?

YNYNY

YNNYN

YYYYY

NNNNN

OTHER ENV. FATE STUDIES RECEIVED Stability in soil, Additional remarks

ECOTOXICOLOGY

4.14.24.34.5.24.6.14.6.24.6.3

Acute toxicity to FishAcute toxicity to DaphniaToxicity to AlgaeChronic toxicity to DaphniaToxicity to Soil dwelling organismsToxicity to Terrestrial plantsToxicity to Birds

YYYYYYN

NNNYNN

NNNY?

N/?

YYYNYY

NNNNNN

YYYYYYY

NNNNNNN

OTHER ECOTOX. STUDIES RECEIVED Toxicity to bacteria

TOXICOLOGY

5.1.15.1.25.1.35.45.5

5.65.85.95.11

Acute OralAcute InhalationAcute DermalRepeated DoseGenetic Toxicity in vitro• Gene mutation• Chromosomal aberrationGenetic Toxicity in vivoReproductive ToxicityDevelopment/TeratogenicityHuman experience

YNYY

YYYNYY

YNYN

YNY

NN

Y/N/?N

Y/NN/?

Y/N/??

N/?

N/?N

YNYY

YYY

YY

NNNN

NNN

NN

YYYY

YYYYYY

NNNN

NNNNNN

OTHER TOXICOLOGY STUDIESRECEIVED

Acute tox. - other routes of administration, Skin irritation/corrosion, Eye irritation/corrosion, Skin sensitisation,Carcinogenicity, Immunotoxicity, Cytotoxicity, Metabolism

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SIDS INITIAL ASSESSMENT REPORT

1. IDENTITY

Name (OECD): Vanillin

CAS number: 121-33-5

Molecular formula: C8H8O3

Molecular Weight: 152,14

Structural formula:��

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Other names: 4-hydroxy-3-methoxybenzaldehydepara-vanillinvanillic aldehyde4-hydroxy-m-anisaldehydemethylprotocatechuic aldehyde3-methoxy-4-hydroxybenzaldehydehydroxy-4-methoxy-3-benzaldehyde

Vanillin is a white crystalline material melting at about 81 oC. The purity is generally above 99.0% w/won dried basis. In vanillin obtained from guaiacol, ethyl vanillin can be an impurity comprising up to 250ppm. Calcium stearate is occasionally added (0.5%) to improve flow-ability. Vanillin has a characteristicpleasant smell and taste of vanilla which is reason for its widespread use.

Vanillin has a vapour presssure of 0.33 Pa and saturated air has a concentration of 0.00029 % at 25 oC,corresponding to 18.0 mg/m3. Vanillin is soluble in water and solubility increases with increasingtemperature. At 25 oC the solubility is 10 g/l. Log Pow value ranges from 1.21 to 1.35 (calculated andmeasured) indicating that vanillin is unlikely to bioaccumulate. The pH of a 5 % solution of vanillin inwater is 4.3. The phenol group of vanillin has a pKa value of 7.38. With increasing pH the molecule willloose a proton, become negatively charged and more soluble in water.

2. GENERAL INFORMATION ON EXPOSURE

For the years 1993-95 the estimated world wide production of vanillin was 10,000 tonnes per year, andan annual increase of 2 % is estimated.

Vanillin is mainly used as an additive to food and beverages (60 %), but considerable amounts are usedfor flavour and fragrances (20-25 %), while 5-10 % is used for intermediates for pharmaceuticals.Vanillin is added to a whole range of food and beverage products in concentrations, depending on theproduct category, from 0.00002 % up 0.1 %. As fragrance ingredient for perfumes etc. vanillin is addedat concentrations ranging from 0.005 % to 0.8 %.

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Vanillin occurs widely in nature both as free vanillin and as constituent of larger molecules. It has beenreported to be present in several essential oils and vanilla pods. Vanillin is also present in plants bound tosugar as a glycoside. A precursor of vanillin is also part of the macromolecule lignin, one of the majorconstituents of wood.

Vanillin has been identified in smoke from burning wood, and also in cigarette smoke.

The majority of vanillin is produced from guaiacol, but significant amounts are also made from lignin, aby-product from the wood-pulp industry. Both production processes are closed except from packaging,where some dust might be formed. Maximum occupational level of organic dust in countries where suchlimits have been set is 6 mg/m3 (Germany) or 5 mg/m3 (Norway). There is no specific occupational limitfor vanillin. From the production site there should be minimal waste to water, but minor dry waste to airand deposit will result. The resulting vanillin product is traded and distributed to the food and perfumeindustry in closed containers. Leakage and exposure during transport can happen accidentally, but this isnot considered a problem.

It can thus be concluded that man is exposed to vanillin by ingestion of food and beverages with addedvanillin. Through the use of perfumes and skin care products some vanillin will also be applied topically.Occupational exposure to the industrial product is limited to rather few individuals.

3. ENVIRONMENT

3.1 Environmental Exposure

3.1.1 General Discussion

Level I fugacity calculation, using a six compartment global reference model shows that vanillin will bedistributed mainly to water (98.5%), minor quantities will be distributed to soil solids (1.41%), whilenegligible quantities will be distributed to other compartments and there are no trends to bioaccumulation.This is in agreement with the Log Pow value of 1.23. Compounds with a Log Pow < 3 will not tend tobioaccumulate.

Abiotic degradation of vanillin has been studied as photodegradation and hydrolytic degradation in water.Vanillin has been estimated to be degraded by sunlight in air with a half-life of 4.7 hours. Meaurement ofhydrolysis in water at different levels of pH, indicated slow rates and the hydrolyses did not reach 10 %in any of the pH systems investigated. The compound is thus considered stable in sterile water.

Biotic degradation of vanillin has been studied in soil, and in water after inoculation with Aspergillusterrus, anaerobic sludge or benthic microorganisms from an eutrophic lake.In unamended garden soil, biodegradation was slow, with about 10 % degraded after 4 weeks, howeverafter amending the soil with "active garden soil" a 41 %degradation was achieved after 21 days. Underaerobic conditions in water after inoculation with Aspergillus terrus, 62.5% was degraded after 6 days.Under anaerobic conditions in water after inoculation with anaerobic sludge, 72 % was degraded after 28days. Benthic microorganisms, dependent on a carbon source in the growth medium, were able to growon vanillin as the only carbon source after 6 days of incubation. It is concluded from these studies thatvanillin is readily biodegradable.

The biochemical oxygen demand (BOD5) and chemical oxygen demand (CODCr) of vanillin has beendetermined. Values of 1.26 mg/mg and 1.76 mg/mg respectively gives an aerobic degradation during 5days incubation at 20 oC of BOD/COD = 72 %. This qualifies vanillin as a compound which is readilybiodegradable.

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The degradation of vanillin by soil invertebrates has been studied by injecting 14C-labelled vanillin into 20specimens of each of an isopod (Osniscus asellus), millipede (Pseudopolydesmus serratus), slug(Deroceras teticulatum), snail (Oxychilus draparnaldi) and earthworm (Eisenia foetida). Of the injectedvanillin; 9-14 % was oxidized to 14C-CO2 after 6 days. At this sample point 2-10 % of non-metabolisedand 13 - 48 % of metabolised vanillin was present in animal tissue, while 1-4 % of non-metabolised and22-66 % of metabolised materials were found in egesta (sand and faeces). Mortality for the differentspecies ranged from 0 to 20 %. It can be concluded from the study that vanillin is readily metabolised inthe tested species. Significant amounts are emitted as CO2, large amounts are excreted as vanillinmetabolites, while only a minor fraction of unmetabolised vanillin is recovered from excretion products orfrom the animals.

Vanillin ingested by man and other mammals is metabolised prior to excretion.

It can be concluded from the degradation experiments that vanillin is susceptible to photodegradation inair, is rather stable to hydrolysis in water, but is readily biodegradable under aerobic conditions. The thestudy of biodegradability under anaerobic conditions shows that vanillin also degrades rapidly underthese conditions. A study in soil invertebrates shows that vanillin is metabolised by all five species tested.

3.1.2 Predicted Environmental Concentration

Monitoring data on vanillin in the environment is limited, but measurements have been done in fog-waterin an area polluted by smog from firewood burning. Content in fog-water was measured during a twomonths period during the winter season in a residential area, 8 measurements ranged between 2 µg/l and 51µg/l (mean 27µg/l). No vanillin was detected in an area with normal activity (no burning). Air samples taken50 m from burning of fruit trees had a concentration of between 7 and 21 µg/l.

In a monitoring well close to an industrial site a concentration of 54 mg/l was found.

It is assumed that emission of vanillin is highest close to production plants. Emission data has beengathered from a plant with an annual production of about 2000 ton/y. This is thought to represent a largeproduction unit category. Shown in the Table below are the estimated and measured emissions for thisproduction plant.

Emission table for a large vanillin production plant. Emissions are either measured or calculated.Production is continuously throughout the year.

Recipient system Secondary treatment Emission based on: Emissionkg/day

-Soil (deposition ofdust)

none estimates 0.15

-Sewage water sewage treatment measurement/estimated 1.15-Surface water none intermittent release

concentration in river<100 ppm, estimated

2.3

-by-product burnt at 600-700 oC measurement/estimated 150

As the frequency and magnitude of intermittent release directly to surface water is not known, it isassumed that this release is continuos. A dilution factor of 10 is assumed from STP to river. The STP isassumed to receive water from 10000 people equivalents each with an output of 200 l/day. It is reportedthat 65 % of vanillin going into STP is degraded. Applying these emission rates as input to the EUSES

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model, gave the following local PECs. PECaquatic = 0.0219 mg/l, PECsediment = 0.0234 mg/kg wwt, PECsoil

= 0.00197 mg/kg wwt. PECsewage plant = 0.219 mg/l.

3.2 Effects on the Environment

3.2.1 Aquatic effects

FishThe acute toxicity of vanillin to fish has been tested in Fathead Minnow (Pimephales promelas). Thefollowing LC50 values were observed for the following observation periods: 1 hr; 173 - 370 mg/l, 24 hr;100 - 131 mg/l, 48 hr; 68.3 - 123 mg/l, 72 hr; 57 - 123 mg/l and 96 hr; 57 - 123 mg/l. It was observedthat fish stopped schooling, became hypoactive, swam at the surface and lost equilibrium prior to death.There are no data available on the chronic toxicity to fish.

DaphniaThe acute toxicity to aquatic invertebrates has been tested in daphnia. After 24 hours exposure, the EC50

value was reported to be 180 mg/l.

A 21 day reproduction study has been performed on daphnia, according to OECD 202 and GLP. After anexposure for 13 days to 100 mg/l, immobilisation occurred in all animals. The EC50 for immobilisation inthe period 13 to 21 days exposure was estimated to 75 mg/l. The reproductive function was howevermore sensitive to vanillin, and after 21 days exposure the EC50 (reproduction) was 16 mg/l (24 mg/l),NOEC 5.9 mg/l (10 mg/l) and LOEC 10 mg/l (18 mg/l) (nominal concentrations given in parentheses).

AlgaeThe toxicity of vanillin to algae was tested in a screening study in a variety of species. Only oneconcentration level (2 mg/l) was used. The green algae Scenedesmus obliquus and Chlorella variegatashowed reduced growth after 3 days but no effect was observed after 7, 14 and 21 days exposure. Whenthe diatoms Gomphonema parvulum and Nitzschia palea were tested for 2 mg/l of vanillin, the formershowed no growth after 3 days exposure and reduced growth after 7, 14 and 21 days. The latter wasunaffected after 3 days, but showed reduced growth after 7 days, however growth was normalised after14 and 21 days. No effect was seen with the blue-green algae Cylinderospermum licheniforme andMicrocystis aeruginosa. These results could indicate that green algae and diatoms are the most sensitivealgae, but since this was a screening test using only one dose level, one should not attach too muchimportance to this finding.

In another report, 50 % growth inhibition of Chlorella vulgaris was observed after 80 hr exposure to 152mg/l. After 160 hr exposure to this concentration the growth inhibition was 30 %. No effects wereobserved at 15 mg/l and 1.5 mg/l.

ConclusionTo summarise the effects of vanillin to the aquatic environment, fish, bacteria and most algae seemsmoderately sensitive to vanillin. There are indications that some algae species are more sensitive tovanillin, and the reproductive function of daphnia was shown to be sensitive. A PNECaquatic based on theNOEC value (5.9 mg/l) found in the Daphnia reproduction test would give a value of 0.059 mg/l (onlyone long term NOEC, application factor=100). A PNEC value based on most sensitive EC50/LC50 value(LC50 fish = 57 mg/l) and an application factor of 1000 give a PNECaquatic of 0.057 mg/l.

MicroorganismsAnother study tested the effect of vanillin on bacteria, yeast and blue-green algae in water. The effect ofvanillin on anaerobic methane formation from sludge was measured, and 49 hr incubation with aconcentration of 1,800 mg/l reduced the methane production with 50 %. Photobacterium phosphoreum

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was however seen to be more sensitive to vanillin, and after 5 minutes incubation, an EC50 value of 58mg/l was observed.

Yeast (Saccharomyces cerevisiae) showed an EC50 value of 179 mg/l after 210 minutes incubation withvanillin.

Conclusion microorganismsPhotobacterium phosphorum being the most sensitive organism and using an application factor of 10gives a PNECSTP of 5.8 mg/l.

3.2.2 Terrestrial effects

PlantsToxicity of vanillin to plants has been tested with lettuce, wheat and cotton. Germination testing withlettuce using water with 650 ±30 mg/l produced a 50 % reduction in germination compared to controls.Using a Petri dish bioassay, the elongation of cotton radicels was visually observable inhibited (11 %)after addition of 30 mg vanillin/dish, while no effect was observed on the wheat.

EarthwormThe effect of vanillin has been tested on growth and survival of the earthworm Eisenia foetida. Thetested concentrations in soil, were 0, 0.1, 1.0, 4 and 8 %, the exposure period was 42 days. Of these, 4%was the lowest concentration which significantly reduced growth rate and caused death (in 80 % of theworms), the NOEC value was 10 g/kg drw.

ConclusionAs plants seem more sensitive than eartworm the EC50 value is multiplied with an application factor of1000, giving a PNECterr of 0.647mg/kg wwt.

3.2.3 Other effects

The white root fungi, which are responsible for breakdown of most of the lignin in nature, are rathertolerant to vanillin, showing 0-33 % inhibition at 152 mg/l, 76-100% at 760 mg/l and no growth at 1520mg/l.

3.3 Initial Assessment for the Environment

With only a few production sites in Europe a regional or contintal exposure assessement does not seemrelevant for this fairly nontoxic compound. A local scenario with a production plant in the large scalecategory should ensure that any possible environmental risks are covered. In the Table below based onsuch a local scenario no PEC/PNEC are found with values above 1. One may therefore conclude thatrisks associated from industrial production of vanillin and from consumer products is very limited. Wastefrom packaging of the raw material, waste of food and use of detergents, soaps and perfumes with addedvanillin account for an added environmental exposure, however this gives a very disperse emission. Seenin relation to observed effect levels in the range of environmental species tested, no hazard to theenvironment can be identified.

Summary table of PEC/PNEC founds for a local scenario for alarge vanillin production plant.Scenario PEC/PNECSurface water 0.385Agricultural soil 0.003Sediment 0.474

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Local STP 0.038

Most monitoring values are for the air compartment, the origin of this vanillin have all been connected toburning of wood. As vanillin is quite soluble in water, exposure in air is not thought to be of importance.A rather high value (54 mg/l) found in water in a monetoring well close to an industrial plant is clearlyabove the PNEC established here for aquatic organisms. In conclusion open air burning of wood mayrepresent a more important source of vanillin exposure in the environment than industrial production.

4. HUMAN HEALTH

4.1 Human Exposure

4.1.1 Occupational exposure

The industrial production of vanillin is a closed process. However, the milling of the dried crystals andthe filling of the product into containers generate some dust to which a limited number of workers areexposed. Also some dust formation is expected in the food and beverage industry during addition ofvanillin.

A study was carried out in the packaging section at Borregaard EuroVanillin, Norway, to assess theinhalation exposure of the operators to vanillin dust. The work in the packaging section is divided among12-18 persons, meaning 2-3 persons per shift. On average, these workers are exposed to vanillin for 1 to1.5 hours per day. For the rest of the day they conduct other operations where they are not exposed tovanillin. The exposure was measured by a constant flow sampler (2 l air/min) carried by the operatorduring the exposure period. The total amount of dust collected corresponded to air concentrations of 4.2,4.3 and 6.0 mg/m3 for the three consecutive days. From microscopic examination of the dust particles,only 10 % of the dust particles were considered to be vanillin.

Occupational exposure to vanillin other than from dust, is of little relevance. For the risk assessment, it isanticipated that occupational exposure is maximum 5 mg/m3. Assuming a person has a respiratoryminute volume (RMV) of 20 l and an exposure for 6 hours per day at the limit concentration, will give adaily exposure by inhalation of 36 mg. For a 70 kg person this makes 0.5 mg/kg/day.

Absorption of vanillin by inhalation of dust will also depend on particle size. Sifting analysis ofcrystalline vanillin revealed that less than 1 % of the particles were smaller than 53 microns. Since onlyparticles smaller than 5 microns are respirable, much less than 1 % of inhaled vanillin can be absorbed.This is valid for vanillin produced from both lignin and guaiacol.

4.1.2 Consumer exposure

The majority of industrially produced vanillin is ingested in the form of food and beverages. Minoramounts are applied topically as skin care products, perfumes etc. The global use of vanillin in food andbeverages imply that almost every human globally is exposed to minute amounts of vanillin by ingestion.The individual doses and exposure can vary due to eating habits and preferences. An Acceptable DailyIntake (ADI) of 10 mg/kg has been agreed between FAO/WHO and EU. For a 70 kg person the ADI is700 mg vanillin which as an example corresponds to minimum 700 g chocolate, or 7000 g of ice cream.For the risk assessment it is assumed that even persons with a high intake of vanillin containing food andbeverages do not have a vanillin intake above the ADI.

4.1.3 Indirect exposure via the environment

Human exposure from the environment is negligible.

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4.2 Effects on Human Health

a) Mode of action of the chemical, toxicokinetics and metabolism

Metabolism studies in rats have shown that vanillin is metabolised to a number of urinary products,primarily vanillic acid, in both free and conjugated forms. Only minor amounts of unmetabolised vanillinis excreted. One person who ingested 100 mg vanillin excreted 96 mg as vanillic acid (94% of the dose)in the next 24 hour period.

b) Acute toxicity

The acute toxicity studies conducted with vanillin are summarised in the following table.

Acute toxicity studies with vanillin

Species, strain No Administration Endpoint Value (mg/kg)

Rat, Sprague Dawley(GLP)

40 Oral, gavage LD50 3925-3976

Rat, Sprague Dawley 40 Oral, gavage LD50 4200

Rat, Sprague Dawley 15 Oral, gavage LD50 3300

Rat, Osborn Mendel 10 Oral LD50 1580

Rat - Oral - 2000

Rat, albino 25 Oral LD50 3830

Guinea Pig 10 Oral LD50 1400

Rat, Sprague Dawley (GLP) 10 Dermal, paste LD0 ³2000

Rabbit 3 Dermal LD50 ³5010

Rat, Sprague Dawley - Intraperitonal LD50 1160

Mouse - Intraperitonal LD50 780

Mouse - Intraperitonal LD50 475

Guinea Pig - Intraperitonal LD50 1190

Rat, albino 50 Subcutaneous LD50 2600

Dog - Intravenously LDL0 1320

The acute toxicity (LD50) of orally administered vanillin to rats seems, when taking into account the morerecent and properly conducted studies, to be in the range of 3500 - 4000 mg/kg. When administeredintraperitonally, intravenously or subcutaneously, the toxicity seems somewhat higher, while littletoxicity has been seen after dermal application. No data are available on the acute toxicity of inhaledvanillin.

c) Repeated dose toxicity

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Many repeated dose toxicity studies have been carried out with vanillin in several animal species. Noneof the studies have been carried out recently, and none have been carried out according to GLP, but someof them are well conducted and hold a high scientific standard. The different studies are summarised inthe following table.

Repeated dose toxicity studies conducted with vanillin.

Species,strain

No Dur-ation

Adm.strat.

Doses End-point

Value (unit)

Rat, O-M 20 27weeks

Oral,feed

1000 ppm(50 mg/kg/day)

NOEL ³1000 ppm(50 mg/kg/day)

Rat, O-M 20 16weeks

Oral,feed

10,000 ppm(500 mg/kg/day)

NOEL ³10,000 ppm(500 mg/kg/day)

Rat, O-M(males)

15 1 year Oral,feed

20,000/50,000 ppm(1000/2500 mg/kg/day)


Rat 80 91 days Oralfeed

3000/10,000/50,000 ppm(150/500/2500 mg/kg/day)

NOEL

LOEL

³3000 ppm(150 mg/kg/day)³10,000 ppm(500 mg/kg/day)

Rat,males

40 26weeks

Oral,feed

1000/5000/10,000 ppm(50/250/500 mg/kg/day)


Dog 8 26-27weeks

Oral,caps

0, 25, 100 mg/kg/day NOEL ³100 mg/kg/day

(O-M: Osborn-Mendel)

The repeated oral administration studies in rat suggest that the NOEL can be as high as 50,000 ppm(2500 mg/kg/day). One oral feed study for 91 days did, however report a NOEL ³ 3000 ppm (150mg/kg/day) and a LOEL ³10,000 ppm (500 mg/kg/day). This study is unpublished, but the informationhas been taken from a citation in 1963. The design and the details of the study are unknown, thus, theother studies seem more reliable. For the assessment, the NOEL of ³50,000 ppm in the 1 year study isused. No particular toxicity has been observed in dogs after repeated oral administration.

No information has been obtained on toxicity after repeated administration through other routes ofexposure.

d) Reproduction developmental toxicity

There are no studies conducted according to the OECD-guidelines, with the aim of assessing the potentialtoxicity of vanillin on the reproductive system of mammals. Most of the repeated dose toxicity studiesconducted in different animal species have, however, included both macroscopic and microscopichistopathological evaluation of the reproductive organs with no reported effect of the test substance.Also, throughout the many years of wide use of vanillin, there are no indications that vanillin is toxic tothe reproductive system.

In a mouse spot test designed to measure the antimutagenic effect of vanillin, pregnant mice were given 3successive oral administrations of vanillin at 125-500 mg/kg at 0, 4 and 24 hours after the injection of themutagen ethylnitrosourea. Vanillin was also given to females in the control group (not given themutagen). Vanillin was shown to have an antimutagenic effect. Even if this study was designed foranother purpose, it indicates that administration of vanillin has no toxic effect on the mouse embryos.

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In another study to test the antimutagenic activity of vanillin, the substance was injected intraperitonallyas a single dose of 50 mg/kg to mice at the 11th day of gestation, either with or without the mutagen. Onday 18 of gestation, dams were killed and examined with their foetuses. It was concluded that the effectof vanillin was comparable to that of saline control with respect to the number of live foetuses, foetalbody weight, foetal mortality and incidence of external and skeletal abnormalities.

Vanillin turned out to be non-teratogenic when tested in the developing chicken embryo test. Fourexposure conditions were used; injection via the air cell and via the yolk and at two different time points(0 and 96 hr). For each condition and at each of five dose levels 100 embryos were treated. All embryosand hatched chicks were examined grossly for any structural or functional abnormality. The highest dosetested was 10 mg vanillin/egg. When injecting the test substance into the air-cell at 0 hr, the observedLD50 value for the embryo was 0.82 mg/egg.

e) Genetic toxicity

Testing of the potential genotoxicity of vanillin has been conducted in a whole range of tests in bacteria(in vitro), in mammalian cells (in vitro) and in different in vivo test. The results of these testing aresummarised in the following 3 tables.

Testing of mutagenicity in tests utilising bacteria.

Test/species strain

No. ofstrains

Concentration range(mg/plate)

Cytotoxicity(mg/plate)

Genotoxicity

+ S9 - S9 + S9 - S9

Ames test/S. typhimurium

(GLP)

5 100 - 5000 > 5000 > 5000 - -


(GLP)

5 50 - 5000 > 5000 > 5000 -


4 100 - 10.000 > 10,000 > 10,000 - -


5 0.5 - 5000 5000 5000 - -


4 456 > 456 > 456 - -


6 £10,000 > 10,000 > 10, 000 - -


2 0.05 - 1000 > 1000 > 1000 - -


2 0 - 1520 > 1520 -

Rev. mutation assay/E. coliS. typhimurium

2 50 - 150 -

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Rev. mutation assay/E. coli WP2

1 0 - 4560 > 4560 -

Rev. mutation assay/E. coli PQ37

1 15 - 1500 > 1500 -

DNA-repair/E. coli K12/PJ5

2 0 - 1520 > 1520 -

DNA-repair/E. coli K12

1 500 (plasm.recomb.)600 (UV-survival)

> 600 -

Testing of potential mutagenicity for vanillin in Ames test, DNA repair test and reverse mutation assayswith E. coli are all negative both in the presence and the absence of a metabolic activation system (S9).Few signs of cytogenicity have been observed, even with doses up to 10 mg/plate.

In vitro mutagenicity testing in mammalian cells.

Test/Cell type

Concen-tration

Observedcytotoxicity

Observed genotoxicity

Cytogenicity test/Human lymphocytes

0 - 612 mg/l Without metabolicactivation,cytotoxicity observedat 612 mg/l

Increased number of aberrationsincluding gaps at 612 mg/l

Cytogenicity test/Mouse fibroblasts

0 - 1224 mg/l Without metabolicactivation,cytotoxicity observedat 1224 mg/l

Vanillin at concentrations 153 - 918mg/l induced multinuclearmutations in the fibroblasts

DNA-repair assay/Chinese Hamsterovary cells - K1

1.5 - 15 mg/l Negative. No chromosomalaberration observed. Vanillinantimutagenic

Gene mutation assay/CHO-Cells

(GLP)

250-1500 mg/l(- S9)183-2440 mg/l(+ S9)

-S9: Completecytotoxicity at 1700mg/l, Cell cycle delayat 509 mg/l.+S9: 2440 ml/lsevere cytotoxicity.

Negative for chromosomalaberrations except at 2440 mg/lwith S9.

Gene mutation assay/Chinese Hamsterfibroblast cells

£1000 mg/lNo metabolicactivation

No cytotoxicity Negative.

Gene mutation assay/Chinese HamsterV79 cells

0-15 mg/l No cytotoxicity Negative.Vanillin showed antimutagenicactivity on UV and X-ray inducedmutations

Gene mutation assay/Chinese HamsterB241 cells

0.05 - 1000mg/plate

No cytotoxicity,neither in presence orabsence of S9

Negative in presence and absence ofS9

Sister chromatid 0 - 50.6 mg/l No cytotoxicity No sister chromatid exchange or

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exchange assay/CHO cells

chromosomal aberration

Sister chromatidexchange assay/Human lymphocytes

114 and 152mg/l

Positive.Significant induction of SCE whenfirst tested at 152 mg/l andconfirmed positive when retested at114 and 152 mg/l

Sister chromatidexchange assay/Human lymphocytes

153, 306 and612 mg/l

Positive effect of vanillin on SCE at153 and 306 mg/l. Positive onchromosomal aberrations at 612mg/l when gaps are included.Negative when gaps are excluded

(CHO: Chinese Hamster Ovary)

Testing of vanillin mutagenicity in mammalian cells in vitro have shown positive effects in some testsystems. Increased number of aberrations has been seen at high vanillin concentrations in humanlymphocytes only when gaps are included. The biological significance of gaps are, however, debated andis not alone any proof of genotoxicity. In mouse fibroblasts, vanillin has been shown to inducemultinuclear mutations. In two independent studies in human lymphocytes, vanillin induced sisterchromatid exchange. High concentrations of vanillin is cytotoxic to mammalian cells. The mutagenicitytesting in vitro in mammalian cells indicates a genotoxic potential of vanillin. However, several of thesestudies were performed at high, unphysiological concentrations that could lead to false positives.

Genetic toxicity testing of vanillin in vivo

Test No Doses Result

MouseMicronucleusAssay

10female/group

500 mg/kg x 21000 mg/kg x 2

Negative.No significant increase in micronuclei inerythrocytes.


3 male/group

125 - 500 mg/kg x 8or500 mg/kg x 2

Vanillin did not induce micronuclei.When vanillin was administered incombination with a positive control(mitomycin C), vanillin decreased itsmutagenic effect.


4 male/group

250 - 500 mg/kg x 7or 500 mg/kg x 8

Vanillin did not induce micronuclei.Vanillin reduced the micronuclei formationinduced by X-rays.

Mouse spot test about 30pregnantfemales/group

125 - 500 mg/kg x 3 Vanillin showed no mutagenic effect, butreduced the mutagenic effect induced by ENU(ethylnitrosourea)

Ring-X loss testin Drosophilamelanogaster

Adultfemaleflies

0.5 - 2 % vanillin in5 % sucrose

Vanillin was seen to have a suppressor effecton ring-X loss that occurs spontaneously inDrosophila melanogaster.

The testing for genetic toxicity of vanillin in vivo is negative in all systems tested and gave no indicationof any genotoxicity.

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f) Carcinogenicity testing

Carcinogenicity testing conducted with vanillin

Test Type No Dosing Duration

Result

Oral feedingcarcinogenicitystudy in the rat

12 maleand 12females/group

Daily in feed, 0,5000, 10,000 and20,000 ppm (250,500 and 1000mg/kg/day)

2 years Negative.Vanillin not a carcinogen.No toxicity observed

Pulmonary tumourtest in mice

15 maleand 15female/group

150 mg/kg or 750mg/kg 3 x weeklyintraperitonalinjections for 8 weeks

8 weeks+ 16weeksobservation

Negative.Vanillin not a carcinogen

Tumour initiatingactivity andcocarcinogeniceffect on mouseskin

18 10 thrice weeklyapplications of 0.3 mlof 20 % vanillin inacetone followed by18 weeklyapplications of 0.3 mlcroton oil

Totally21weeksobserva-tionperiod

No significant increase in localtumours. No cocarcinogeniceffect

Short term cellproliferation of ratstomach

5/group

Daily combinedadministration of 2 %vanillin ± 0.3 %NaNO2 in drinkingwater.

4 weeks No significant effect onoesophagus and on theforestomach mucousa. Inglandular stomach, thickness andlabelling indices significantlyincreased by the combination.

Anti-photocarcinogenicproperties ofvanillin in mice

3/group

0.5% vanillin in diet 40weeks

Vanillin did not reduce tumourlatency but significantly reducedtumour multiplicity (- 48 %)

A full 2 years oral feeding carcinogenicity study has been conducted in rats with a negative result. Therewas no indication of vanillin being an experimental carcinogen. The other tests conducted confirm thisfinding and some tests even indicate that vanillin reduces the tumourgenicity of carcinogenic treatments.

None of the carcinogenicity studies presented have been conducted according to GLP, but the 2 yearsstudy (conducted prior to GLP regulations) seem to hold a high scientific standard and includes anappropriate number of animals and groups. Less emphasis should be put on the other studies mentioned,but they support the conclusions from the 2 years study.

g) Other data

Skin irritation testing with vanillin

Test type No Dosing Result

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Rabbit skin irritationtest

6 Application of groundedsample for 24 hr

No irritation

Closed patch test inGuinea Pigs

5 1, 2, 5 or 10 % vanillin appliedfor 48 hours

All observations were negative,there were no signs of irritation

The two skin irritation studies were both negative.

Skin sensitisation testing with vanillin


Open Epicutaneoustest (OET) in GuineaPigs

6 - 8 0.03 - 30 % and undilutedvanillin applied on 2 cm2 ofclipped flank skin. Read after14 hr. Repeated administrationfor 21 days

Minimum irritation concentration(30 %) after 1 application, but 3% after 21 applications

Closed patch test inGuinea Pigs

10 10 % Vanillin applied for 24hours 3 times a week for 2weeks and then read after 1, 24and 48 hours

All observations were negative,there were no signs ofsensitisation or allergy in any ofthe animals.

Draize test in GuineaPigs

6 - 8 Day 0: 0.05 ml of 0.1 %solution in saline.Day 1-9: 0.1 ml of 0.1%.Challenged again on days 35and 49 with 0.05 ml

No allergic effects were seen inGuinea Pigs with vanillin.

Maximisation Test inGuinea Pigs

6-8/group

Intradermal injection of 0.1 ml5% vanillin ±Freunds completeadjuvance

Vanillin showed positive allergiceffects in Guinea Pigs

Freunds completeadjuvance test inGuinea Pigs

6-8 0.05 ml of undiluted vanillinmixed with 0.05 ml Freundscomplete adjuvance andinjected intradermally into theneck on days 0, 2, 4, 7, and 9

Vanillin showed positiveallergenic effects in Guinea Pigs

Freunds completeadjuvance test inGuinea Pigs

10 10 % in acetone Weak sensitive

Maximisation Test inGuinea Pigs (GLP)

40 17.5 - 35 % in ethanol, 73 % inpaste

The test article did not provokeany reaction or cutaneoussensitisation in any of theanimals examined


21 50 % vanillin Positive result in 60 % ofanimals


- 10 - 50 % vanillin Positive

Mouse ear swelling 15 - 50 % vanillin in ethanol No sensitisation

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test 25

In the skin sensitisation testing with vanillin, 5 out of 10 tests showed positive results indicating thatvanillin has an allergenic potential. The other tests were negative, including the only test conductedaccording to GLP.

Eye irritation testing


Rabbit eye irritationtest

6 55 mg sample of finelygrounded powder, 24 hoursexposure

Slightly irritating. Graduallyimproving from 48 - 120 hr.After 168 hr, all scored zero.

Only vanillin powder was irritating to the eye due to mechanical stress from crystals.

Immunotoxicity testing of vanillin


Immunotoxicity inmice

30/group

250, 500 and 1000 mg/kgintragastrically daily for 5days

No signs of immunotoxicity

Test forimmunosuppression invitro

50 mg/culture.Test for plaque-forming cellresponse

Vanillin stimulated plaqueforming cell response up to 300%, indicating animmunostimulatory effect

Immunotoxicity in anin vitro antibodyproducing assay

Vanillin, 200 mg/culture.Anti sheep red blood cells

Vanillin suppressed the in vitroanti sheep red blood cell antibodyresponse indicting an immunesuppressing effect.

Immunomodulatoryeffect in vitro

1, 10, 100, 300, 1000 mg/ml.Microbicidal activity of mousemacrophages tested

Vanillin is a weak modulator ofmacrophage function

Testing of possible immunotoxicity indicated that vanillin is not immunotoxic, but one study indicated animmunostimulating effect, while another indicated an immunosuppressive effect.

h) Human data

Vanillin was tested in the closed patch test on healthy normals and on subjects with dermatoses. In thetest with the healthy volunteers, test substance was applied on the back (20% concentration) of 29subjects for 48 hr or at the upper inside of the arm (2 % concentration) of 30 subjects for 24-72 hr.Vanillin (0.4 % concentration) was applied for 24 -48 hr on the upper inside of the arm of 35 subjectswith dermatoses. Both in the healthy persons and in those with dermatoses vanillin was negative in alltests. No sign of irritation or erythema was observed.

A patch test was carried out on 30 workers in a vanillin factory and compared with 15 controls. Abouthalf of the workers had dermatitis. Vanillin was applied undiluted and removed after 48 hours. No signsof irritation or sensitisation was observed with vanillin.

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Vanillin (2 %) was tested in the Maximisation test, where the skin area is made permeable by applyingsodium lauryl sulphate for 24 hr prior to 48 hr repeated application of the test material. Vanillin wasnegative for all 25 subjects tested and concluded to be non-sensitising in humans.

In two different studies, positive reactions to vanillin have been reported in patients who already weresensitised to Balsam of Peru. From these studies vanillin was considered to be a secondary allergen.

In a double blind challenge test, an asthmatic patient was given vanillin by inhalation at 1.5 hr intervals,two or three times, providing no reaction occurred within 15 minutes of challenge. There was someevidence that vanillin reduced lung function at oral doses of 0.24 and 1 mg. Itching of the ears and throatwas also described.

4.3 Initial Assessment for Human Health

In the human health risk assessment the consumer exposure (by oral intake) is taken as the ADI value(10.0 mg/kg/day).

Occupational exposure has been identified as inhalation of vanillin dust by operators in the packagingarea of the factory. This exposure has been quantified to a maximum of 0.5 mg/kg/day. This level islikely to never be reached since the amount of vanillin in the total dust is estimated to only about 10 %.Additionally, much less than 1 % of the product has a particle size small enough to reach the lungs.

Skin exposure to vanillin has not been quantified, but both consumers and workers will be subject to aminor extent of such exposure.

Assessment from acute and repeated dose toxicityFrom the acute toxicity studies, the LD50 value for rats (3500 - 4000 mg/kg) gives a safety margin of 350- 400 for consumers, and this is considered satisfactory.

From the oral repeated dose toxicity studies a NOEL of 2500 mg/kg/day was observed after oral feedingto rats. This gives a safety margin of 250 for consumers use, and this is considered acceptable.

Assessment from reproduction and development studiesThe potential toxicity of vanillin to the reproductive system has been studied in several connections.Microscopic and macroscopic histopathological evaluation of reproductive organs were performed inconnection with repeated dose studies. In the mouse spot test and in a study to test the possibleantimutagenic effect of vanillin, it was given to pregnant mice. The potential teratogenicity of vanillin hasalso been tested in the developing chicken embryo test. There has been no sign of vanillin being toxic tothe reproduction system, or to the developing embryo in any of these tests. Even though areproduction/development study carried out according to OECD-guidelines is lacking, the present use andproduction of vanillin indicate no such risk.

Assessment from genotoxicity and cancer studiesThe testing of the potential genotoxicity of vanillin is comprehensive. Mutagenicity testing in bacteriawas negative. Testing in vitro in mammalian cells gave positive results in tests for sister chromatidexchange and chromosomal aberration in human lymphocytes. These results indicate that vanillin undercertain testing conditions might be genotoxic. However, in vivo genotoxicity tests were negative. Anoverall evaluation of the test results indicate that vanillin is not likely to pose a genetic risk to humans.

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The carcinogenicity studies, including a 2 years study in rats, did all give negative results. It is concludedthat the present use and production of vanillin represent little risk for genotoxicity and there are no signsof carcinogenicity.

Assessment from skin irritation and sensitisation testingSkin irritation and sensitisation testing have shown that in some tests vanillin turned out to be a sensitiserand thus a potential allergen. These results are, however, not conclusive and the negative human datasupport the opinion that vanillin is not a human allergen.

5. CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

Only minute quantities of vanillin are released from the vanillin production sites. Some occupationalexposure to vanillin as inhalation of dust has been identified. The consumers will be directly exposed tovanillin as most of the produced material is ingested as flavour additive to food and beverages.

From a distribution model, it seems that vanillin in the environment will be distributed to the aqueouscompartment, and there is no trend to bioaccumulation. The emissions of vanillin from the vanillinproduction and from the consumer products to the environment, are undetectable and represent no hazard.

During the present reviewing of the environmental and human safety data for vanillin, no particular riskhas been identified which should give reason to concern or additional toxicity testing in animals.

The use of vanillin as a food additive is approved by authorities world wide, and FDA has granted GRASstatus to its use. This is in agreement with the experience with vanillin in consumer products during manyyears without any confirmed report of adverse events.

5.2 Recommendations

It is recommended to make efforts to obtain more data on the occupational exposure through monitoringof inhalation of vanillin dust in contaminated working areas. Such monitoring data should also includeparticles larger than those normally considered respirable.

6. REFERENCES

References are not given in the SIDS initial assessment report. It is referred to those given in the FullSIDS Dossier.

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SIDS PROFILESIDS Dossier

DATE: 20.08.96

1.01 A. CAS No. 121-33-5

1.01 C. CHEMICAL NAME(OECD Name)

Vanillin

1.01 D. CAS DESCRIPTOR Not applicable

1.01 G. STRUCTURAL FORMULA ��

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OTHER CHEMICALIDENTITY INFORMATION

1.5 QUANTITY Estimated global production in 1993 was 10,000tons.

1.7 USE PATTERN (a) Wide dispersive use as foodstuff additive tofood and food essences industry (0.005-0.1%).(Vanilla sugar 2%).

(b) Non dispersive use as intermediate forindustrial synthesis of pharmaceuticals.

(c) Wide dispersive use as odour agent incosmetics for personal and domestic use (usual:0.005-0.01%, max: 0.03-0.1%).

(d) Wide dispersive use as odour agent in perfumesfor personal and domestic use (usual: 0.2%, max:0.8%).

1.9 SOURCES AND LEVELSOF EXPOSURE

1. Amount released from production site(EuroVanillin, Norway) to soil: < 100 kg/year.

2. Amount released from production site(Borregaard EuroVanillin, Norway) to water: <1500 kg/year.

3. Exposure through food products: 0.005-0.1% infoods containing Vanillin.

4. Exposure through cosmetic products (perfumes,creams, lotions, soap, detergents): 0.001-0.8% incosmetics containing Vanillin.

ISSUES FORDISCUSSION(IDENTIFY,IF ANY)

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1. GENERAL INFORMATION

1.01 SUBSTANCE INFORMATION

A. CAS-Number: 121-33-5B. Name (IUPAC): 4-hydroxy-3-methoxybenzaldehydeC. Name (OECD): VanillinD. CAS Descriptor: Not applicableE. EINECS-Number: 204-465-2F. Molecular Formula: C8H8O3

G. Structural Formula:

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H. Substance Group: Not applicableI. Substance Remark:J. Molecular Weight: 152.14

1.02 OECD INFORMATION

A. Sponsor Country: Norway

B. Lead Organisation: Norwegian Pollution Control AuthorityContact Person: Marit KopangenAddress: P.O.Box 8100 Dep.

N-0032 OSLONORWAYTel: +47 22 57 34 00Fax: +47 22 67 67 06

C. Name of Responder: Borregaard EuroVanillinAddress: P.O.Box 162

N-1701 SARPSBORGNORWAYTel: +47 69 11 80 00Fax: +47 69 11 86 40

1.1 GENERAL SUBSTANCE INFORMATION

A. Type of Substance: Element ( ); Inorganic ( ); Natural substance ( ); Organic ( x ); Organometallic ( ); Petroleum product ( )

B. Physical State

(at 20oC and 1.013 hPa):Gaseous ( ); Liquid ( ); Solid ( x )Crystalline

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C. Purity: > 99.0% w/w on dried basis

1.2 SYNONYMS

para-VanillinVanillic aldehydeVanillaldehyde4-hydroxy-m-anisaldehydemethylprotocatechuic aldehydeProtocatechualdehyde-3-methyl ether3-methoxy-4-hydroxybenzaldehydehydroxy-4-methoxy-3-benzaldehyde

1.3 IMPURITIES

CAS No: 121-32-4EINECS No: 204-464-7Name: Ethyl Vanillin (3-ethoxy-4-hydroxybenzaldehyde)Value: < 250 ppmRemarks: In vanillin obtained from guaiacol only, and not in vanillin from lignin

raw material.Valid for EuroVanillin KS, Norway.

1.4 ADDITIVES

CAS No: 1592-23-0EINECS No: 216-472-8Name: Calcium stearateValue: Approx. 0.5%Remarks: Is added only occasionally (one single product) to improve flowability.

Valid for EuroVanillin KS.

1.5 QUANTITY

Remarks: Estimated global production:1993: 10,000 metric tonnes. (Divided on 2 big (>2000 metric tonnes/year) and 5-8 smaller producers.) 1

1994: 10,000 metric tonnes 2

1995: 10,000 metric tonnes 2

Reference: 1) Project Euromarketing, 1993.2) Estimated 2% increase, by EuroVanillin KS, Norway.

1.6 LABELLING AND CLASSIFICATION

A. Labelling

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(a)Type: Directive 67/548/EECSpecific limits:Symbols:Nota:R-phrases:S-phrases:Text of S-phrases:Remarks: No labelling required (no dangerous properties)

(b)Type: Other: Directive 88/388/EEC.Remarks: Requirements:

- Name of producer- "Aroma" or "Vanillin"- For food- Nature identical flavouring- Identification of the lot- Weight/VolumeThis is valid for Vanillin sold for manufacturing of food products.

B. Classification

Type: Directive 67/548/EECCategory of danger:R-phrases:Remarks: No classification required (no dangerous properties)

1.7 USE PATTERN

A. General

(a)Main category: Wide dispersive useIndustrial category: Other: Food and food essences industryUse category: Food/foodstuff additivesRemarks: Vanillin was given GRAS (Generally Recognized As Safe) status by

FEMA (Flavor and Extract Manufacturers' Association) in 1965 and isapproved by the FDA for food use (GRAS). The Joint FAO/WHOExpert Comittee on Food Additives (1967) has published a monographand specifications for vanillin, giving an unconditional ADI (AcceptableDaily Intake) of 0-10 mg/kg. The Council of Europe (1974) listedvanillin, giving it an ADI of 10 mg/kg (Opdyke, 1977).

(b)Main category: Non dispersive useIndustrial category: Chemical industry: synthesisUse category: Intermediate for pharmaceuticals

(c)Main category: Wide dispersive use

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Industrial category: Personal and domestic useUse category: Odour agent for cosmeticsRemarks: Added to cosmetics by industry

(d)Main category: Wide dispersive useIndustrial category: Personal and domestic useUse category: Odour agent for perfumesRemarks: Added to perfumes by industry

(e)Remarks: Vanillin is used as pharmaceutic aid (flavour), as flavouring agent in

confectionary, beverages, foods, as a odour agent in perfumery and asreagent in analytical chemistry (The Merck Index, 1989). Besides beinga very popular flavour in the food industry, vanillin is also useful in thesynthesis of drugs; 40% of the vanillin is consumed in manufacturingdrugs such as Aldomet (antihypertensive drug), L-dopa (treatment ofParkinson's disease) and Trimethoprim (treatment of upper respiratory-tract infections and some strains of venereal disease). Vanillin is alsoused in the perfume and metal-plating industries. Other uses for vanillininclude prevention of foaming in lubrication oils, as a brightener in zinccoating baths, as an activator for electroless plating of zinc, as an aid tothe oxidation of linseed oil, as an attractant in insecticides, as an agent toprevent mouth roughness caused by smoking tobacco, in the preparationof syntans for tanning, as a solubilizing agent for riboflavin and as acatalyst to polymerize methyl methacrylate (Kirk-Othmer, 1983).

(f)Remarks: Estimated global Vanillin consumption by end use application:

Food & Beverages: 60%Flavour and Fragrances: 20-25%Pharmaceutical intermediates: 25%

Reference: Garshol, 1996.

B. Uses in Consumer Products

(a)Function: Amount present: Physical state:

Flavouring agent in foods and beverages: Dissolved or dispersed

Non-alcoholic beverages 0.0063% 1

Ice cream, Ices etc. 0.0095% 1

Candy 0.020% 1

Baked goods 0.022% 1

Gelatines and puddings 0.012% 1

Chewing gum 0.027% 1

Syrups 0.033-2.0% 1

Chocolate 0.097% 1

Toppings 0.015% 1

Margarine 0.00002% 1

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Bonbons up to 0.050% 2

Chocolate, plain up to 0.055% 2

Chocolate milk up to 0.030% 2

Fondants up to 0.015% 2

Fudge up to 0.055% 2

Marshmallow up to 0.040% 2

Nougat up to 0.055% 2

Vanillin sugar 2% 2 Powder

Fragrance ingredient for perfumes: 0.2% usual Dissolved0.8% max 3

Fragrance ingredient for creams, lotions: 0.005% usual Dissolved0.03% max 3

Fragrance ingredient for detergents: 0.001% usual Dissolved0.01% max 3

Fragrance ingredient for soap: 0,01% usual Dissolved0,1% max 3

Reference: 1) Furia et al, 1975, Ash, 1995.2) ACF ChemieFarma NV.3) Opdyke, 1977

(b)Food category Level of use (weighted mean), ppm

Usual MaximumBaked goods 74.5 106.0Break cereals 353.0 353.0Fats, oils 96.0 100.0Sweet sauce 358.0 363.0Gelatine pudding 47.7 117.0Snack foods 200.0 200.0Beverage type I 39.4 97.4Milk products 221.0 314.0Frozen dairy 26.7 55.2Meat products 1.5 2.7Soft candy 247.0 408.0Beverage type II 30.1 47.1Hard candy 26.4 193.0Chewing gum 1.5 445.0Miscellaneous 468.0 2090.0

Reference: FEMA, 1985.

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1.8 OCCUPATIONAL EXPOSURE LIMIT VALUES

A. Exposure limit value

(a)Type: Norwegian Administrative NormValue: 5 mg organic dust/m3 (average through 8 hours)

No particular limit for Vanillin.Reference: Direktoratet for Arbeidstilsynet, 1995.

(b)Type: MAK (DE)Value: 6 mg dust/m3

No particular limit for Vanillin.Reference: List of MAK and BAT Values, 1995.

(c)Type: BAT (DE)Value: No values for neighter Vanillin nor organic dustReference: List of MAK and BAT Values, 1995.

(d)Type: TRK (DE)Value: Not relevant. TRK is for carcinogenic and mutagenic substances only.Reference: List of MAK and BAT Values, 1995.

(e)Type: OEL (EEC)Value: Neighter Vanillin nor organic dust has been treated yet.Reference: Directive 91/322/EEC, 1991.

(f)Type: COSHH (UK)Value: 10 mg total inhalable dust/m3 (8-hour TWA)

5 mg respirable dust/m3 (8-hour TWA)No particular limit for Vanillin.

Reference: Health and Safety Executive, 1996.

(g)

Remarks: Percent in "saturated air"at 25oC, 760 mmHg: 0.00029 % = 2.9 ppm1.Conversion factors in air: 1 mg/l = 161 ppm; 1 ppm = 6.2 mg/m3 2.

Reference: 1) Clayton et al, 1993, 2) HSDB, 1994.

B. Short time exposure limit value

Value: No short time exposure limit value (Norway)Length of exposure period:Frequency:Remarks:Reference: Direktoratet for Arbeidstilsynet, 1995.

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1.9 SOURCES OF EXPOSURE

(a)Source: Media of release: From air in the packing area to soil.

Quantities per media: < 100 kg/yearRemarks: Valid for EuroVanillin KS site, Norway (see 1.5 QUANTITY for

information regarding estimated number of producers).The whole process is closed, except from the packing station.Some Vanillin is lost due to raise of dust in the packing area.When cleaning the packing area, the Vanillin dust is treated as ordinary organic waste, and is being deposited at a public landfill.

Reference: Estimation done by EuroVanillin, 1995.

(b)Source: Media of release: Discharge to water.

Quantities per media: < 500 kg/yearRemarks: Valid for Borregaard EuroVanillin site, Norway (see 1.5 QUANTITY

for information regarding estimated number of producers).The raffinate from K1800 in factory 2 is transferred to the biological treatment plant, where at least 65% of the Vanillin in the raffinate is decomposed.

Reference: Estimation done by Borregaard EuroVanillin, 1996.

(c)Source: Media of release: Discharge to water.

Quantities per media: < 1000 kg/yearRemarks: Valid for Borregaard EuroVanillin site, Norway (see 1.5 QUANTITY

for information regarding estimated number of producers).Although the process is closed, there are from time to time waterdischarged to the river that contains Vanillin, but in concentrations ofless than 100 ppm.

Reference: Estimation done by Borregaard EuroVanillin, 1996.

(d)Source: Media of release: From raffinate to by-product.

Quantities per media: < 150 mt/yearRemarks: Valid for Borregaard EuroVanillin site, Norway (see 1.5 QUANTITY

for information regarding estimated number of producers).The various raffinate and tar streams from both plants are collected,evaporated and sold to Scandinavian craft mills for make-up.

The make-up product are burnt at temperatures around 600-700oC,and Vanillin is decomposed to CO2 and water, so practically no

Vanillin is released.Reference: Estimation done by Borregaard EuroVanillin, 1996.

(e)Source: Media of release: Food products

Quantities per media: 0.005-0.1%Remarks:Reference: 1.7 USE PATTERN B. Uses in Concumer Products (this SIDS)

(f)

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Source: Media of release: Cosmetic products (perfumes, creams,lotions, soap, detergents)

Quantities per media: 0.001-0.8%Remarks:Reference: 1.7 USE PATTERN B. Uses in Concumer Products (this SIDS)

(g)Remarks: Waste from end products: Information not available.

Occupational exposure: Dust (inhalation).Occurence: Vanillin occurs widely in nature. It hasbeen reported in the essential oil of Java Citronella, in benzoin, Perubalsam, and clove-bud oil, and chiefly in Vanilla pods. Vanillin is alsopresent in the plants as glucose and vanillin. Anothersource of vanillin isthe lignin from the wood-pulp industry. (BIBRA Toxicity Profile, 1990.Opdyke, 1977).

Vanillin occurs in nature as a glycoside, which hydrolyses to vanillinand sugar. It has been identified in many oils, balsams, resins andwoods. The best known source of vanillin is the vanillin plant (VanillaPlanifolia), a member of the orchid family (Kirk-Othmer, 1983)

Vanillin occurs naturally in vanilla, in potato parings, and in Siambenzoin (The Merck Index, 1989).

Vanillin occurs in balsams and is a constituent of lignins (Parke, 1968).

Vanillin occurs in tissues of certain plants, crude beet sugar, asparagus,and Asafetida (Osol et al, 1975).

Vanillin occurs in essential oil of Java Citronella and in Clove bud oil(Furia et al, 1975).

Vanillin has been identified in over 40 food products, in fruits (berries,urrants up to 0.02 ppm), in asparagus (1.2 ppm), in spices (mint,nutmeg), crispbread, alcoholic beverages (at levels varying fro 0 to 9.6ppm), coffee, tea and vanilla. The highest concentration, up to 23.000ppm, has been found in vanilla (Feron et al, 1991).

Vanillin has been found in anise, asparagus, Balsam Peru, roasted, barley, beer, brandy, bread volatiles, rye crispbread (0.14 ppm), grape musts, maple sap, maple syrup, evaporated milk, heated milk, dried mushroom, yellow passion fruit, popcorn, smoked belly pork (23.4 ppm), rum, jamaican rum (0.25 ppm), spearmint oil, tomato, vanilla extract (200-3100 ppm, 1000-2400 ppm, 250-1880 ppm), Bourbon vanilla pods, rice vinegar, whiskey, wine (FEMA, 1985).

(h)Value: Day 1: 4.2 mg/m3 total dust (0.42 mg/m3 Vanillin, estimated)

Day 2: 4.3 mg/m3 total dust (0.43 mg/m3 Vanillin, estimated)Day 3: 6.0 mg/m3 total dust (0.60 mg/m3 Vanillin, estimated)

Length of exposure period: Day 1: 2 hoursDay 2: 1.25 hoursDay 3: 1 hour

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Frequency: The above exposure periods were the total exposure to Vanillin dust for the personnel during the actual days of working. The packaging operation was divided between 2-3 operators, so it is not nessesarily the same person who was exposed all the three days (see remarks).

Remarks: The operators are exposed to Vanillin dust at the packaging section only, i.e. the filling operation. The rest of the production process is closed.The work in the packaging section is divided between a 12-18 persons (6 shifts, 2-3 persons per shift). The packaging operation counts for approximately 3 hours per shift per day. This means that each operator is exposed to Vanillin dust 1-1.5 hour per day on average, or more likely 3 hours 2-3 days per week. The rest of the time they are also carrying out other operations of which they are not exposed to Vanillin dust.The test was carried out during the packaging of Vanillin ex lignin at Borregaard Eurovanillin, Norway.A pump carried by the operator was used (Du Pont constant flow sampler S-2500). 2 liters air/min.Filters: Closed filter holders. Filters of cellulose acetate, diameter: 37 mm, mesh opening 0.8 microns. The filter was exposed in the zone of breathing.Microscopic evaluation showed that most of the dust particles seemed to be dust from outside the location. The gates were open during the exposure periods.The Vanillin concentration is estimated to be max. 10% of the total dust.

Reference: Ramberg, 1996.

Remarks: Sifting analysis of Vanillin samples:Vanillin ex lignin: 1.0 % < 53 micronsVanillin ex guaiacol: 1.1% < 53 micronsMean value of 4 parallel samples.Method: 3 g sample, LP3 Sonic Sifter Separator (ATM Corporation), pulse at amplitude 5, 10 minutes, US Standard Sieves (ATM Corporation).

Reference: Pettersen, 1996.

1.10 ADDITIONAL REMARKS

A. Options for disposal

Remarks: Sweep, scoop or vacuum up all spilled material, contaminated soil and other contaminated material and place in clean, dry container for disposal. The material may be incinerated in a suitable chemical incinerator.If there are arrangements for treatment of effluents on site, wash down with water to sewer.

B. Other remarks

Remarks: Water pollution factors1: Reduction of amenities:

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- T.O.C. in water: 0.2 ppm4 ppm

- Detection: 0.06 mg/l0.2 mg/kg

- Recognition: 4.0 mg/kg

Odour detection/recognition2:- Recognition in water: 4.00 ppm- Detection in water: 0.20 ppm- Detection in air: 1.10x10-8 ppb- Detection in air: 2.00x20-4 ppb

Odour detection may be different by orders of magnitude because methods for making these determinations are totally subjective andscales used in these evaluations are not standardized. Additionally, the olfactory receptors may accomodate very rapidly to odours, resulting in inconsistent human responce.

Reference: 1) Verschueren, 1983.2) Clayton et al, 1993

2. PHYSICAL-CHEMICAL DATA

2.1 MELTING POINT

(a)

Value: 80-81 oCDecomposition: Yes ( ) No ( x ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Lewis, 1992, HSDB, 1994.

(b)

Value: 80-81oC (81-83 oC)Decomposition: Yes ( ) No ( ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: The Merck Index, 1989.

(c)

Value: 80-82 oCDecomposition: Yes ( ) No ( ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:

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Reference: Numata et al, 1992.

(d)

Value: 81-82 oCDecomposition: Yes ( ) No ( ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Inoue et al, 1981.

(e)

Value: 81-83oCDecomposition: Yes ( ) No ( x ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Hawley, 1971, Kirk-Othmer, 1983, Sigma-Aldrich, 1988, Food

Chemicals Codex, 1994, The United States Pharmacopeia 23/TheNational Formulary 18, 1995.

(f)

Value: 81-84 oCDecomposition: Yes ( ) No ( ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: Capillary methodGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: British Pharmacopeia, 1993, Deutsches Arzneibuch, 1994.

(g)

Value: ca. 82 oCDecomposition: Yes ( ) No ( x ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Rhône-Poulenc, 1994.

(h)

Value: 82-83 oCDecomposition: Yes ( ) No ( ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Di Biase et al, 1979.

(i)

Value: 82-83.5 oC

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Decomposition: Yes ( ) No ( x ) Ambigous ( )Sublimation: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Clayton et al, 1993.

2.2 BOILING POINT

(a)

Value: 127 oCPressure: 1.33 hPaDecomposition: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Kirk-Othmer, 1983.

(b)

Value: 170 oCPressure: 20 hPaDecomposition: Yes ( ) No ( ) Ambigous ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Kirchhof et al, 1934, Sigma-Aldrich, 1988, The Merck Index, 1989,

Weast et al, 1989.

(c)

Value: 285 oCPressure: 1013 hPaDecomposition: Yes ( x ) No ( ) Ambigous ( )Method: The temperature at which the liquid phase is in equilibrium with the

vapour at a pressure of 760 mmHg (101.325 kPa)*.GLP: Yes ( ) No ( ) ? ( x )Remarks: When heated to decomposition vanillin emits acrid smoke and irritating

fumes **.Reference: Susuki et al, 1978, Verschueren, 1983, Weast et al, 1989, The Merck

Index, 1989, Lewis, 1992 (**), Clayton et al, 1993, Lide, 1994-1995(*).

2.3 DENSITY

(a)Type: Bulk density ( ); Density ( x ); Relative density ( )Value: 1.056 g/cm3

Temperature: 20 oCMethod: No dataGLP: Yes ( ) No ( ) ? ( x )

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Remarks:Reference: Kirk-Othmer, 1983, Verschueren, 1983, The Merck Index, 1989,

Weast et al, 1989, Lewis, 1992, Clayton et al, 1993, Lide, 1994-1995, Rhône-Poulenc, 1994.

(b)Type: Bulk density ( x ); Density ( ); Relative density ( )Value: 400-900 kg/m3

Temperature: Room temperatureMethod: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks: Varying because of different crystal sizes.Reference: EuroVanillin KS, 1995b, Rhône-Poulenc, 1994.

2.4 VAPOUR PRESSURE

(a)Value: 0.0022 hPa

Temperature: 25oCMethod: Calculated ( ); Measured ( )

No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Clayton et al, 1993.

(b)Value: 0.17 hPa


No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Rhône-Poulenc, 1994.

(c)Value: 1.33 hPa


No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Verschueren, 1983.

(d)Value: 9.73 hPa


No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:

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Reference: Rhône-Poulenc, 1993.

(e)Value: 13.3 hPa



(f)Value: 133 hPa

Temperature: 214.5oCMethod: Calculated ( ); Measured ( )


2.5 PARTITION COEFFICIENT log10Pow

(a)Log Pow: 1.21Temperature: No dataMethod: Calculated ( ); Measured ( )

No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Holmes, et al, 1976, LOGP and Related Parameters Computerized

Database, 1987, Rhône-Poulenc, 1994.

(b)Log Pow: 1.23

Temperature: 22oCMethod: Calculated ( ); Measured ( x )

GC-analysis of saturated o/w in equilibrium.GLP: Yes ( ) No ( x ) ? ( )Remarks: The compound is slightly dissociative.Reference: EuroVanillin KS, 1993.

(c)Log Pow: 1.21-1.35Temperature: No dataMethod: Calculated ( x ); Measured ( x )GLP: Yes ( ) No ( ) ? ( x )Remarks: Different experimental and estimation methods.Reference: Bodor, N. et al, 1989.

(d)Log Pow: 1.29-1.33

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Temperature: No dataMethod: Calculated ( x ); Measured ( )

OtherGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Leo, 1978.

(e)Log Pow: 1.31Temperature: No dataMethod: Calculated ( ); Measured ( )

No dataGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Korenman et al, 1975.

2.6 WATER SOLUBILITY

A. Solubility

(a)Value: 3 g/l

Temperature: 4.4oCDescription: Miscible ( ); Of very high solubility ( ); Of high solubility ( );

Soluble ( ); Slightly soluble ( ); Of low solubility ( ); Of very low solubility ( ); Not soluble ( )

Method: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Mange et al, 1924.

(b)Value: 5.2 g/l

Temperature: 15.6oCDescription: Miscible ( ); Of very high solubility ( ); Of High solubility ( );

Soluble ( ); Slightly soluble ( ); Of low solubility ( ); Of very lowsolubility ( ); Not soluble ( )

Method: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Mange et al, 1924.

(c)Value: 9 g/l

Temperature: 23.9oCDescription: Miscible ( ); Of very high solubility ( ); Of High solubility ( );


Method: No dataGLP: Yes ( ) No ( x ) ? ( )

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Remarks:Reference: Mange et al, 1924.

(d)Value: 10g/l

Temperature: 25oCDescription: Miscible ( ); Of very high solubility ( ); Of High solubility ( );

Soluble ( ); Slightly soluble ( x ); Of low solubility ( );Of very lowsolubility ( ); Not soluble ( )

Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: The Merck Index, 1989, Food Chemicals Codex, 1994, The United

States Pharmacopeia 23/The National Formulary 18, 1995.

(e)Value: 50 g/l



Method: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks:Reference: Tieman, 1877.

(f)Value: 50 g/l



Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Food Chemicals Codex, 1994, The United States Pharmacopeia

23/The National Formulary 18, 1995.

(g)Value: 62.5 g/l



Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: The Merck Index, 1989.

(g)Value:Temperature:

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Description: Miscible ( ); Of very high solubility ( ); Of High solubility ( );Soluble ( ); Slightly soluble ( x ); Of low solubility ( ); Of very lowsolubility ( ); Not soluble ( )

Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Lide, 1994-1995.

B. pH Value, pKa Value

(a)pH Value: 4.3Concentration: 5% in water, saturated solution

Temperature: 25oCMethod: Measured by electrode.GLP: Yes ( ) No ( x ) ? ( )pKa Value:Remarks:Reference: EuroVanillin KS, 1995a.

(b)pH Value:Concentration:Temperature:Method:GLP: Yes ( ) No ( x ) ? ( )

pKa Value: 7.38 at 25oCRemarks: pKa value: Measured; spectrophotometric, solvent: water.Reference:. Robinson et al, 1955.

2.7 FLASH POINT

Value: 153oCType of test: Closed cup ( x ); Open cup ( ); Other ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Rhône-Poulenc, 1993, Rhône Poulenc, 1994.

2.8 AUTO FLAMMABILITY

Value: > 400oCPressure: No dataMethod: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Rhône-Poulenc, 1994.

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2.9 FLAMMABILITY

Results: Extremely flammable ( ); Extremely flammable - liquified gas ( ); Highly flammable ( ); Flammable ( ); Non flammable ( ); Sponaneously flammable in air ( ); Contact with water liberates highly flammable gases ( ); Other ( x )Dust explosivity hazard in air.

Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Rhône-Poulenc, 1994.

2.10 EXPLOSIVE PROPERTIES

Results: Explosive under influence of a flame ( ); More sensitive to friction than m-dinitrobenzene ( ); More sensitive to shock than m-dinitrobenzene ( ); Not explosive ( ); Other ( x ) Dust explosivity hazard in air under influence of ignition sources1.

Method: No dataGLP: Yes ( ) No ( x ) ? ( )Remarks: Can react violently with Br2, HClO4, potassium-tert-butoxide, tert-

chlorobenzene + NaOH, formic acid + thallium nitrate2.Reference: (1) Rhône-Poulenc, 1993.

(2) Lewis, 1992.

2.11 OXIDIZING PROPERTIES

Results: Maximum burning rate equal or higher than reference mixture ( ); Vigorous reaction in preliminary test ( ); No oxidizing properties (x );Other ( )

Method: No dataGLP: Yes ( ) No ( ) ? ( x )Remarks:Reference: Rhône-Poulenc, 1994.

2.12 OXIDATION: REDUCTION POTENTIAL

Not Applicable

2.13 ADDITIONAL DATA

A. Partition co-efficient between soil/sediment and water (Kd)

Value: No dataMethod:GLP: Yes ( ) No ( ) ? ( )Remarks:Reference:

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B. Other data

(a)Results: Freely soluble in ethanol, methanol 1,2,3,4,5, chloroform 1,2,3, carbon

disulfide, glacial acetic acid and pyridine 1.Soluble in ether 1,2,3,4,5, aceton , benzen 3 and oils 1

Dissolves in dilute solutions of alkali hydroxides 1,4,5.Remarks:Reference: (1) The Merck Index, 1989.

(2) Food Chemicals Codex, 1994.(3) Lide, 1993-1994.(4) British Pharmacopeia, 1993.(5) Deutsches Arzneibuch, 1994.

(b)

Results: Decomposes above 160oC; differential thermal analysis on crude Vanillin before purification showed slightly exothermic decomposition

at 130-200oC and strongly exothermic decomposition at 300oC.Remarks:Reference: Rhône-Poulenc, 1988.

(c)Results: Slowly oxidizes somewhat when exposed to moist air, affected by

light.Remarks:Reference: The Merck Index, 1989, HSDB, 1994.

(d)

Results: Incompatibilities with aluminium above 100oC, with strong oxidizing and reducing agents, with strong bases or alkalis. Decomposition products: phenol (pyrolysis), CO and CO2.

Remarks:Reference: Sigma-Aldrich, 1988, Rhône-Polenc, 1994.

(e)Results: Exposure to air causes Vanillin to oxidize slowly to vanillic acid.

When Vanillin is exposed to light in an alcoholic solution, a slow dimerization takes place, with the formation of dehydrovanillin.

Remarks:Reference: Kirk-Othmer, 1983.

3. ENVIRONMENTAL FATE AND PATHWAYS

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3.1 STABILITY

3.1.1 PHOTODEGRADATION

Type: Air ( x ); Water ( ); Soil ( ); Other ( )Light source: Sun light ( x ); Xenon lamp ( ); Other ( )Light spectrum: Average daylightRelative intensity: Leads to 1.5 x 106 OH-radicals per cm3

Spectrum of substance:Concentration of substance:

Temperature: 25oCDirect photolysis:

Half life:Degradation:Quantum yield:

Indirect photolysis:Type of sensitizer: OH-radicalsRate constant (radical): 27.3 x 10-12 cm3 molecule-1 sec-1

Degradation: Half-life: 4.7 hoursMethod: Calculated according to Atkinson (AOPWIN, Version 1.70, June

1995)GLP: Yes ( ) No ( x ) ? ( )Test substance: VanillinRemarks: Validity of Estimation: 91% of estimations are within a factor of two

of the experimental value.Reference: Völkel, 1995.

3.1.2 STABILITY IN WATER

Type: Abiotic (hydrolysis) ( x ); Biotic (sediment) ( )Half life:Degradation: StableMethod: OECD TG 111: "Hydrolysis as a function of pH" 1981.GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1-1.2

Purity: 99.9%Remarks: The study was terminated after the preliminary test, since the

hydrolysis did not reach > 10% in any of the pH-systems.Reference: Semb et al, 1996.

3.1.3 STABILITY IN SOIL

Type: Field trial ( ); Laboratory ( x ); Other ( )Radiolabel: Yes ( ); No ( x ); ? ( )Concentration: 1190 mg/kg

Soil temperature: 25oCSoil humidity: 7 g water/100 g soil dry weightSoil classification: DIN19863 ( ); NF X31-107 ( ); USDA ( ); Other ( x )

Natural soil amended with 9% montmorillonite

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Content of clay etc.: Clay 9%, Silt 34%, Sand 57%Organic Carbon: 5.8%Soil pH: 5.6Cation exchange capacity: 8.2 meq/100 g soil dry weightMicrobial biomass:Dissipation time:Dissipation: 41% after 21 daysMethod: Other: Carbon dioxide evolution measurements.GLP: Yes ( ) No ( ) ? ( x )Test substance: VanillinRemarks: As no significant amounts of CO2 were detected after 1 week of

incubation, a suspension of active "garden soil" was inoculated into each soil in an attempt to increase the number of organisms capable of utilizing Vanillin. The soils were then incubated for another 3 weeks.Other results:No clay added (pH 5,1): 9% biodegradation.9% koaolinite added (pH 5,0): 11% biodegradation.

Reference: Bewley et al, 1984.

3.2 MONITORING DATA (ENVIRONMENT)

(a)Type of measurement: Background ( x ); At contaminated site ( x ); Other ( )Media: Fog water and interstitial airResult: Among other compounds (guaiacol, syringol and their derivatives)

Vanillin concentrations were found as:- On residental areas of Davis (Dec. 1990- Feb. 1991):14. Dec.: 47 ug/l18. Dec.: 44 ug/l4. Jan.: 51 ug/l5. Jan.: 29 ug/l7. Feb.: 2 ug/l8. Feb.: 6. 31 and 8 ug/l (3 samples)- On agricultural areas (Jan. 1991):8. Jan.: Not detected (2 samples)11. Jan.: 20. 7 and 17 ug/l (3 samples)

Remarks: Vanillin evolved from wood burning.Fog water and interstitial air samples were collected on two sites in California:- City of Davis, population 50,000, in residental area, to determine theambient concentrations of wood smoke markers.- Kearney Agricultural Research Center, typical agricultural area in the Central Valley:8. Jan. 1991: ambient samples with nonusual activities.11. Jan. 1991: while a waste pile of orchard purnings (primarily peach and almond) was burning, approx. 50 m from the sampler, to determine the local impact of such a waste disposal.

Air samples, air samples filters and water filters were extracted by sonication with 3 aliquots of acetone for 30 minutes each. After

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combination over anhydrous Na2SO4 and reduction by rotary evaporation, gas chromatography was performed.

Reference: Sagabiel et al, 1993.

(b)Type of measurement: Background ( ); At contaminated site ( x ); Other ( )Media: Ground waterResult: 54 mg/lRemarks: Water collected from a contaminated monitoring well located at

Nuclepore Corporation facility in Pleasanton, California.Reference: California Regional Water Quality Control Board, 1983.

3.3 TRANSPORT AND DISTRIBUTION BETWEEN ENVIRONMENTALCOMPARTMENTS INCLUDING ESTIMATED ENVIRONMENTALCONCENTRATIONS AND DISTRIBUTION PATHWAYS

3.3.1 TRANSPORT

Type: Adsorption ( ); Desorption ( ); Volatility ( ); Other ( )Media:Method:Result:Remarks: No dataReference:

3.3.2. THEORETICAL DISTRIBUTION (FUGACITY CALCULATION)

Media: Air-biota ( ); Air-biota-sediment-soil-water ( x ); Soil-biota ( ); Water-air ( ); Water-biota ( ); Water-soil ( ); Other ( )

Method: Fugacity level I ( x ); Fugacity level II ( ); Fugacity level III ( ); Fugacity level IV ( ); Other (calculation) ( ); Other (measurement)( )Generic Models for Evaluating the Regional Fate of Chemicals (Mackay et al, 1992).

Result: The potential environmental distribution of Vanillin was calculated using a generic level I fugacity model. The distribution was:Water: 98.5%Air: 0.066%Soil solids: 1.41%Sediment soilds: 0.031%Suspended sediments: 0.00098%Fish: 0.000080%

Remarks: The calculation was based on a half-life of 4.7 hr in air and 1500 hr in water and soil.

Reference: Dahl, 1996.

3.4 IDENTIFICATION OF MAIN MODE OF DEGRADATION IN ACTUAL USE

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Result: No dataRemarks:Reference:

3.5 BIODEGRADATION

(a)Type: Aerobic ( x ); Anaerobic ( )Inoculum: Adapted ( x ); Non-adapted ( )

Aspergillus sp. (Aspergillus terreus)Concentration of the chemical:Related to COD ( ); DOC ( ); Test substance ( )Medium: Water ( ); Water-sediment ( ); Soil ( ); Sewage treatment ( x )Degradation: 62.5% after 6 daysResults: Readily biodeg. ( x ); Inherently biodeg. ( ); Under test condition no

biodegradation observed ( ); Other ( )Method: Assays in 5 litre batch reactor, steady flow of air 720 ml/min, stirring

rate 200 rpm.

Incubation at 28 oC for 6 days.GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin in the waste produced by the olive-oil extraction industry.Remarks: Biodegradation of Vanillin in the waste produced by the olive-oil

extraction industry.Reference: Martinez et al, 1993.

(b)Type: Aerobic ( ); Anaerobic ( x )Inoculum: Adapted ( x ); Non-adapted ( )

Anaerobic sludgeConcentration of the chemical:300 mg/l related to COD ( ); DOC ( ); Test substance ( x )Medium: Water ( x ); Water-sediment ( ); Soil ( ); Sewage treatment ( )

Containing (NH4) 2PO4, NH4Cl, MgCl2-6H2O, KCl, MnCl2-4H2O,CoCl2-6H2O, H3BO3, CaCl2-2H2O, Na2MoO4-2H2O, ZnCl2, FeCl2-4H2O, NaHCO3, Na2S-9H2O and 1% (v/v) vitamin solution.

Degradation: 0% after 12 days72% after 28 days

Results: Readily biodeg. ( x ); Inherently biodeg. ( ); Under test condition no biodegradation observed ( ); Other ( )

Method: A serum-bottle variation of the Hungate technique for growing anaerobic bacteria was adapted from Miller and Wolin (1974). The

cultures were incubated in the dark at 35oC.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin. No further data.Remarks: 10 methanogenic enrichment cultures were found to degrade Vanillin

after a lag phase of 12 +/- 1.2 days;period of gas production (CO2 and CH4) 16 +/- 1.1 days;conversion of substrate carbon to gas 72 +/- 1.4 days.Vanillin is biodegradable to methane and carbon dioxide under strict anaerobic conditions.

Reference: Healy et al, 1979.

(c)

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Type: Aerobic ( ); Anaerobic ( x )Inoculum: Adapted ( x ); Non-adapted ( )

Benthic microorganisms from an eutrophic lake (Microorganisms unable to grow on a medium without carbon source were picked)

Concentration of the chemical:100 mg/l related to COD ( ); DOC ( ); Test substance ( x )Medium: Water ( ); Water-sediment ( ); Soil ( ); Sewage treatment ( )

Bacto Yeast Nitrogen Base (Difco) without aminoacids.Degradation: From samples of bottom deposites at 2 sites of the eutrophic lake

Jeziorak (Poland), 26% of the microorganisms were able to utilize Vanillin as sole source of carbon after 6 days of incubation.

Results: Readily biodeg. ( ); Inherently biodeg. ( ); Under test condition no biodegradation observed ( ); Other ( x )

Method: After 6 days of incubation at 26oC, colonial development was assessed by comparison of the cultures with control plates containing no carbon source. Plates containing ferrous gluconate (100 mg/l) were used to check the viability of the inoculum.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin. No further data.Remarks:Reference: Strzelczyc et al, 1972, Opdyke, 1977.

3.6 BOD5, COD OR RATIO BOD5/COD

BOD5

Method: ISO 5815 (Water quality - Determination of biochemical oxygen demand after 5 days (BOD5) - Dilution and seeding method. 1989)

Concentration: 2 and 4 mg/l related to COD ( ); DOC ( ); Test substance ( x )Value: 1.26 mg O2/mg

GLP: Yes ( x ) No ( ) ? ( )

CODMethod: ISO 6060 (Water quality - Determination of chemical oxygen demand.

1989)Value: 1.76 mg O2/mg

GLP: Yes ( x ) No ( ) ? ( )

Ratio BOD5/COD: 72 %

Test substance: As prescribed by 1.1 - 1.2.Purity: 99.9%

Remarks: Meets criteria of >50 % degradationReference: Källqvist, 1996a.

3.7 BIOACCUMULATION

Species:Exposure period:Temperature:Concentration:

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BCF:Elimination: Yes ( ) No ( ) ? ( )Method:Type of test: Calculated ( ); Measured ( )

Static ( ); Semi-static ( ); Flow-through ( ); Other ( )GLP: Yes ( ) No ( ) ? ( )Test substance:Remarks: According to Nordisk Miljørapport 1990 substances having log Pow

lower than or equal to 3.0 are not potentially bioaccumulative.Reference: Nordisk Miljørapport, 1990.


A. Sewage treatment

Results: In case of disposal, pure Vanillin can be recirculated, e.g. recrystallized.Do not release product into the environment. Decant and purify polluted waste water before it is released into the drains.Incinerate product in licensed, suitable chemical incinerator, equipped with an after burner and a scrubber.

Remarks:Reference: EuroVanillin KS, 1995c.

B. Other information

Results:Remarks: When Vanillin 5-(14 C), 1 ul was injected in 5 soil invertebrates, 9-

14% were oxidized to 14-CO2 at 15oC over 6 days.Soil invertebrates:- Isopod (Oniscus asellus)- Millipede (Pseudopolydesmus serratus)- Slug (Deroceras reticulatum)- Snail (Oxychilus draparnaldi)- Earthworm (Eisenia foetida)Mortality:- Isopod: 3/20- Slug: 2/20- Snail: 1/20- Earthworm: 2/20Approximately 2-10% of non-metabolized and 13-48% of metabolized Vanillin were present in the animal tissues after 6 days.Correspondingly, 1-4% and 22-66% of these materials were found in egesta (in sand and feces).

Reference: Neuhauser et al, 1978.

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4. ECOTOXICOLOGICAL DATA

4.1 ACUTE/PROLONGED TOXICITY TO FISH

(a)Type of test: Static ( ); Semi-static ( ); Flow-through ( x ); Other ( )

Open-system ( ); Closed-system ( x )Species: Pimephales promelas (Fathead Minnow)Exposure period: 96 hoursResults: LC50 (24h) =

LC50 (48h) =LC50 (72h) = 123 (104-146) mg/lLC50 (96h) = 123 (104-146) mg/lNOEC =LOEC =

Analytical monitoring: Yes ( x ) No ( ) ? ( )Method: US EPA Environmental Research Laboratory - Duluth, MN, USA,

1981. (See Remarks).GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Eastman Kodak Co, Rochester, NY, USA.

Purity: research grade.Remarks: Fresh water.

Duplicate tests with 25 fish per concentration.

Test temperature: 24.7 (SD 0.72) oC.Dissolved oxygen: 6.2 (SD 0.37) mg/l.Hardness: 53.5 (SD 1.00) mg/l CaCO3.Alkalinity: 39.5 (SD 1.00) mg/l CaCO3.Tank volume: 5.5 lMedium renewal: 13.1 l/daypH: 7.00 (SD 0.09)Fish mean length: 17.3 (SD 2.023) mmFish mean weight: 0.055 (SD 0.0182) gFish age: 31 days.Fish loading: 0.2 g/l95% confidence limits.Affected fish lost equilibrium prior to death.

Reference: Brooke et al, 1984.

(b)Type of test: Static ( ); Semi-static ( x ); Flow-through ( ); Other ( )

Open-system ( ); Closed-system ( x )Species: Pimephales promelas (Fathead Minnow)Exposure period: 96 hoursResults: LC50 (24h) = 109.8 mg/l

LC50 (48h) = 63.8 mg/lLC50 (72h) = 57 (53.0-61.3) mg/lLC50 (96h) = 57 (53.0-61.3) mg/lNOEC =LOEC =

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Analytical monitoring: Yes ( x ) No ( ) ? ( )Method: US EPA Environmental Research Laboratory - Duluth, MN, USA,

1980. (See Remarks).GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Eastman Kodak Co, Rochester, NY, USA.

Purity: research grade.Remarks: Fresh water.

Duplicate tests with 25 fish per concentration.

Test temperature: 23.9 (SD 0.73) oC.Dissolved oxygen: 7.2 (SD 0.93) mg/l.Hardness: 51.0 (SD 0.00) mg/l CaCO3.Alkalinity: 40.5 (SD 0.00) mg/l CaCO3.Tank volume: 6.3 l.Medium renewal: 5.7 l/day.pH: 7.16 (SD 0.32).Fish mean length: 20.0 (SD 1.67) mm.Fish mean weight: 0.131 (SD 0.0323) g.Fish age: 29 days.Fish loading: 0.52 g/l.95% confidence limits.Affected fish stopped schooling, became hypoactive, swam at the surface, and lost equilibrium prior to death.

Reference: Brooke et al, 1984.

(c)Type of test: Static ( x ); Semi-static ( ); Flow-through ( ); Other ( )

Open-system ( ); Closed-system ( x )Species: Pimephales promelas (Fathead Minnow)Exposure period: 96 hours

Test 1 Test 2 Test 3Results: LC50(1h) = 348 mg/l >173 mg/l >173 mg/l

LC50 (24h) = 100 mg/l 127 mg/l 125 mg/lLC50 (48h) = 97 mg/l 121 mg/l 116 mg/lLC50 (72h) = 88 mg/l 121 mg/l 116 mg/lLC50 (96h) = 88 mg/l 121 mg/l 116 mg/lNOEC =LOEC =

Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: Static nonrenewal laboratory bioassay.

Test water: Reconstituted water.Replicate tests with 10 fish per concentration.

Test temperature: 18-22 oC.3 litres cylindrical glass battery jars containing 2 litres of test water.Fish length: 11-31 mm.Fish age: 4-8 weeks.Fish acclimated 48 hours in flowing water (test 1 and 2 in Lake Superior water, test 3 in reconstituted water).

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Curtin Metheson Scientific Inc.

Purity: reagent gradeRemarks: Dissolved oxygen measured during the test < 4 mg/l. This normally

invalidates the test in standardised methods. Earlier work has shown

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that at these concentrations low oxygen itself can cause adverse effects on fathead minnows in long-term toxicity tests.Toxicant concentrations are nominal.

Reference: Mattson et al, 1976.

(d)Type of test: Static ( x ); Semi-static ( ); Flow-through ( ); Other ( )

Open-system ( ); Closed-system ( x )Species: Pimephales promelas (Fathead Minnow)Exposure period: 96 hours

Test 1 Test 2Results: LC50 (1h) = >173 mg/l 370 mg/l

LC50 (24h) = 131 mg/l 125 mg/lLC50 (48h) = 123 mg/l 116 mg/lLC50 (72h) = 121 mg/l 112 mg/lLC50 (96h) = 121 mg/l 112 mg/lNOEC =LOEC =

Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: Static nonrenewal laboratory bioassay.

Test water: Lake Superior water.Duplicate tests with 10 fish per concentration.

Test temperature: 18-22 oC.3 litres cylindrical glass battery jars containing 2 litres of test water.Fish length: 11-31 mm.Fish age: 4-8 weeks.Fish acclimated 48 hours in flowing water (test 1 and 2 in Lake Superior water, test 3 in reconstituted water).

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Curtin Metheson Scientific Inc.

Purity: reagent gradeRemarks: Dissolved oxygen measured during the test < 4 mg/l. This normally

invalidates the test in standardised methods. Earlier work has shown that at these concentrations low oxygen itself can cause adverse effects on fathead minnows in long-term toxicity tests.Toxicant concentrations are nominal.

Reference: Mattson et al, 1976.

4.2 ACUTE TOXICITY TO AQUATIC INVERTEBRATES (e.g. DAPHNIA)

A. Daphnia

Type of test: Static ( ); Semi-static ( ); Flow-through ( ); Other ( )Open-system ( ); Closed-system ( )

Species: Daphnia magna (Crustacea)Exposure period: 24 hoursResults: EC50 (24h) = 180 mg/l

EC50 (48h) =ECxx (...h) =NOEC =

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LOEC =Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: ISO 6341 15 "Water quality - Determination of the inhibition of the

mobility of Daphnia magna Straus (Cladocera, Crustacea)".GLP: Yes ( ) No ( x ) ? ( )Test substance: As prescribed by 1.1 - 1.2.

Purity: >= 99.6% w/w.Remarks:Reference: Rhône-Poulenc, 1983.

B. Other aquatic organisms


Species:Exposure period:Results: EC50 (24h) =

EC50 (48h) =ECxx (...h) =NOEC =LOEC =

Analytical monitoring: Yes ( ) No ( ) ? ( )Method:GLP: Yes ( ) No ( ) ? ( )Test substance:Remarks: No data.Reference:

4.3 TOXICITY TO AQUATIC PLANTS (E.G. ALGAE)

(a)Species: Scenedesmus obliquus (green algae)End point: Biomass ( x ); Growth rate ( ); Other ( )Exposure period: 3, 7, 14 and 21 daysResults: EC50 (...h) =

ECxx (...h) =NOEC = 2 mg/l (7, 14 and 21 days)LOEC = 2 mg/l (3 days)Partially toxic after 3 days exposure (growth occured in the presence of Vanillin, but the amount was not as great as that in the control flask).Non-toxic after 7, 14 and 21 days exposure (growth in the presence of Vanillin was similar to that in the control flask).

Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: Static - LAB.

Culture medium approximated Gerloff's modification of Chu No 10 with the amount of nitrate doubled (Palmer and Maloney, 1953).

Tests carried out in 25 ml Erlenmeyer flasks, incubated at 22oC.Sterile, double strength medium with 1/15 to 1/30 by volume of an actively growing culture of the alga.

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7.5 ml of this inoculated medium was combined with a like quantity of distilled water containing 4 ppm by weight of Vanillin (final concentration of 2 ppm Vanillin in normal strength medium, inoculum of 1/30 to 1/60 the volume of the original algal culture).Number of algal cells in the medium at the start of the test: Approx. 125,000 cells/ml.Visible algal growth was recorded at 3, 7, 14 and 21 days and compared with the growth in a control flask containing 15 ml of normal strength inoculated culture medium without Vanillin.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further data.Remarks: Only concentration tested.Reference: Palmer et al, 1955.

(b)Species: Chlorella variegata (green algae)End point: Biomass ( x ); Growth rate ( ); Other ( )Exposure period: 3, 7, 14 and 21 daysResults: EC50 (...h) =




Tests carried out in 25 ml Erlenmeyer flasks, incubated at 22oC.Sterile, double strength medium with 1/15 to 1/30 by volume of an actively growing culture of the alga.7.5 ml of this inoculated medium was combined with a like quantity of distilled water containing 4 ppm by weight of Vanillin (final concentration of 2 ppm Vanillin in normal strength medium, inoculum of 1/30 to 1/60 the volume of the original algal culture).Number of algal cells in the medium at the start of the test: Approx. 125,000 cells/ml.Visible algal growth was recorded at 3, 7, 14 and 21 days and compared with the growth in a control flask containing 15 ml of normal strength inoculated culture medium without Vanillin.


(c)Species: Gomphonema parvulum (diatom)End point: Biomass ( x ); Growth rate ( ); Other ( )Exposure period: 3, 7, 14 and 21 days

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Results: EC50 (...h) =ECxx (...h) =NOEC =LOEC = 2mg/lToxic after 3 days exposure (no growth occured in the presence of Vanillin, but occured in the control flask).Partially toxic after 7, 14 and 21 days exposure (growth occured in the presence of Vanillin, but the amount was not as great as that in the control flask).



Tests carried out in 25 ml Erlenmeyer flasks, incubated at 22oC.Sterile, double strength medium with 1/15 to 1/30 by volume of an actively growing culture of the alga.7.5 ml of this inoculated medium was combined with a like quantity of distilled water containing 4 ppm by weight of Vanillin (final concentration of 2 ppm Vanillin in normal strength medium, inoculum of 1/30 to 1/60 the volume of the original algal culture).Number of algal cells in the medium at the start of the test: Approx. 125,000 cells/ml.Visible algal growth was recorded at 3, 7, 14 and 21 days and compared with the growth in a control flask containing 15 ml of normal strength inoculated culture medium without Vanillin.


(d)Species: Nitzschia palea (diatom)End point: Biomass ( x ); Growth rate ( ); Other ( )Exposure period: 3, 7, 14 and 21 daysResults: EC50 (...h) =




Tests carried out in 25 ml Erlenmeyer flasks, incubated at 22oC.Sterile, double strength medium with 1/15 to 1/30 by volume of an actively growing culture of the alga.7.5 ml of this inoculated medium was combined with a like quantity of distilled water containing 4 ppm by weight of Vanillin (final

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concentration of 2 ppm Vanillin in normal strength medium, inoculum of 1/30 to 1/60 the volume of the original algal culture).Number of algal cells in the medium at the start of the test: Approx. 125,000 cells/ml.Visible algal growth was recorded at 3, 7, 14 and 21 days and compared with the growth in a control flask containing 15 ml of normal strength inoculated culture medium without Vanillin.


(e)Species: Chlorella vulgaris (algae)End point: Biomass ( x ); Growth rate ( ); Other ( )Exposure period: 80 hoursResults: EC50 (80h) = ca. 1 mmol/l

ECxx (...h) =NOEC =LOEC =

Analytical monitoring: Yes ( x ) No ( ) ? ( )Method: OtherGLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Fluka, Buchs, Switzerland.

Remarks: Test temperature: 26oC.Growth inhibition:- after 80h: 50% at 1 mmol/l- after 160h: 30% at 1 mmol/l.

Reference: Dedonder et al, 1971.

4.4 TOXICITY TO BACTERIA

(a)Type: Aquatic ( x ); Field ( ); Soil ( ); Other ( )Species: Anaerobic sludgeExposure period: 49 hoursResults: EC10 (...h) =

EC50 (49h) = 1800 mg/lEC80 (49h) = 1880 mg/lEC100(...h) =

Analytical monitoring: Yes ( ) No ( x ) ? ( )

Method: The methanogenic inhibition was determined at 30oC in standard toxicity assays, utilizing anaerobic granular sludge as inoculum.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin - commercially available.Remarks: 50% and 80% methane production inhibition concentrations.

Specific methanogenic activity measurements were performed in 0.3 dm3 glass serum flasks. Granular sludge (1g VSS (volatile suspended solids)/dm3, from a full scale reactor treating distillery wastewater, notacclimated to the toxicants) was transferred to 0.1 l of the basalmedium, and acetate was added from a neutralized at pH7 stock solution

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to obtain a final concentration of 2 g COD/l. Subsequently, the flasks

were sealed and placed in a reciprocating shaker at 30 +/- 2oC.After 1 day of incubation, the acetate concentration was measured and replenished to obtain 2 g COD/l. The required amount of inhibitorycompound was added.After 2 days of exposure, the acetate concentration was replenished to 2 g COD/l, and the bottles were reincubated for 1 hour prior to the determination of the methane production rate. The methane composition in the head space content of each serum flask was determined periodically during the subsequent 4 to 5 hours.

Reference: Sierra-Alvarez et al, 1991.

(b)Type: Aquatic ( x ); Field ( ); Soil ( ); Other ( )Species: Photobacterium phosphoreum (bacteria)Exposure period: 5 minutesResults: EC10 (...h) =

EC50 (5 min) = 0.38 mmol/lEC100(...h) =

Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: Microtox TestGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin. (Determined according to the standard method defined by

Beckman Instruments Inc. (1982)). No further data.Remarks: Mean of replicate tests.Reference: Cronin et al, 1991.

(c)Type: Aquatic ( x ); Field ( ); Soil ( ); Other ( )Species: Saccharomyces cerevisiaeExposure period: 210 minutesResults: EC10 (...h) =

EC50 (210 min) = 179 mg/lEC100(...h) =

Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: The Yeast TestGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin. Of highest grade purity, checked by chromatographic and

spectrophotometric methods.

Remarks: Test temperature: 30oC.The initial density was adjusted to approx. E+8 cells per ml.The inhibition of the growth rate is evaluated by counting the cell number with a microscope or a Coulter counter.

Reference: Koch et al. 1993.

(d)Type: Aquatic ( ); Field ( ); Soil ( x ); Other ( )Species: White-rot fungiExposure period: 96 hoursResults: EC10 (...h) =

EC50 (...h) =EC100(...h) =

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Vanillin inhibited fungal growth by:- 0-33% at 1 mmol/l- 76-100% at 5 mmol/l- no growth at 10 mmol/l

Analytical monitoring: Yes ( ) No ( x ) ? ( )Method: Fungal growth measurements:

Radial growth was measured at four equidistant points, on plates inoculated in the centre with a 3 mm diam. agar disc taken from the growing edge of each fungal culture

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin. No further data.Remarks: Strains tested 96h:

- Bjerkandera adusta- Coriolus versicolor- Phlebia radiata- Polyporus dichrous- Pycnoporus cinnabarinusStrains tested 42h:- Pleorotus ostreatusStrains tested 30h:- Phanerochaete chrysosporium K3 and PHE3.Fungal growth rate were linear with time.

Reference: Buswell et al, 1994.

(e)Type: Aquatic ( x ); Field ( ); Soil ( ); Other ( )Species: Microcystis aeruginosa (blue-green algae)Exposure period: 3, 7, 14 and 21 daysResults: EC10 (...h) =

EC50 (...h) =EC100(...h) =NOEC = 2 mg/lLOEC =Non-toxic after 3, 7, 14 and 21 days exposure (growth in the presence of Vanillin was similar to that in the control flask).



Tests carried out in 25 ml Erlenmeyer flasks, incubated at 22oC.Sterile, double strength medium with 1/15 to 1/30 by volume of an actively growing culture of the alga.7.5 ml of this inoculated medium was combined with a like quantity of distilled water containing 4 ppm by weight of Vanillin (final concentration of 2 ppm Vanillin in normal strength medium, inoculum of 1/30 to 1/60 the volume of the original algal culture).Number of algal cells in the medium at the start of the test: Approx.125,000 cells/ml.Visible algal growth was recorded at 3, 7, 14 and 21 days and compared with the growth in a control flask containing 15 ml of normal strength inoculated culture medium without Vanillin.

GLP: Yes ( ) No ( x ) ? ( )

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Test substance: Vanillin; no further data.Remarks: Only concentration tested.Reference: Palmer et al, 1955.

(f)Type: Aquatic ( x ); Field ( ); Soil ( ); Other ( )Species: Cylindrospermium licheniforme (blue-green algae)Exposure period: 3, 7, 14 and 21 daysResults: EC10 (...h) =

EC50 (...h) =EC100(...h) =NOEC = 2 mg/lLOEC =Non-toxic after 3, 7, 14 and 21 days exposure (growth in the presence of Vanillin was similar to that in the control flask).



Tests carried out in 25 ml Erlenmeyer flasks, incubated at 22oC.Sterile, double strength medium with 1/15 to 1/30 by volume of an actively growing culture of the alga.7.5 ml of this inoculated medium was combined with a like quantity of distilled water containing 4 ppm by weight of Vanillin (finalconcentration of 2 ppm Vanillin in normal strength medium, inoculum of 1/30 to 1/60 the volume of the original algal culture).Number of algal cells in the medium at the start of the test: Approx. 125,000 cells/ml.Visible algal growth was recorded at 3, 7, 14 and 21 days and compared with the growth in a control flask containing 15 ml of normal strength inoculated culture medium without Vanillin.


4.5 CHRONIC TOXICITY TO AQUATIC ORGANISMS

4.5.1 CHRONIC TOXICITY TO FISH


Species:End point: Length of young fish ( ); Weight of young fish ( );

Reproduction rate ( ); Other ( )Exposure period:Results: EC50 (...d) =

ECxx (...d) =NOEC =LOEC =

Analytical monitoring: Yes ( ) No ( ) ? ( )

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Method:GLP: Yes ( ) No ( ) ? ( )Test substance:Remarks: No dataReference:

4.5.2 CHRONIC TOXICITY TO AQUATIC INVERTEBRATES

Type of test: Static ( ); Semi-static ( x ); Flow-through ( ); Other ( )Open-system ( ); Closed-system ( x )

Species: Daphnia magna (strain A)End point: Mortality ( x ); Reproduction rate ( x ); Other ( )Exposure period: 21 daysResults: Immobilisation of all animals occured at the highest concentration

tested (100 mg/l) within 13 days exposure.No immobilised animals were recorded in the controls and at concentrations up to 56 mg/l.Hence the EC50 value for immobilisation estimated as the geometricmean concentration for 0 and 100% immobilisation was 75 mg/l from 13days to 21 days exposure.

Nominal conc. Measured conc. EC50 (21d) (reproduction) = 24 mg/l 16 mg/lNOEC (reproduction) = 10 mg/l 5.9 mg/l LOEC (reproduction) = 18 mg/l 10 mg/l

Analytical monitoring: Yes ( x ) No ( ) ? ( )Method: OECD TG 202 (1984) "Daphnia sp., acute Immobilisation Test and

Reproduction Test.GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1 -1.2.

Purity: 99.9%Remarks: Weighted mean concentrations of Vanillin in the test medium was

calculated from the measured concentrations as described in the draft revised OECD TG 202 part II (1996).

Reference: Källqvist, 1996b.

4.6 TOXICITY TO TERRESTRIAL ORGANISMS

4.6.1 TOXICITY TO SOIL DWELLING ORGANISMS

Type: Artificial Soil ( ); Filter paper ( ); Other ( x )Species: Eisenia foetida (Earthworm)End point: Mortality ( x ); Weight ( x ); Other ( )Exposure period: 42 daysResults: EC50 (...d) =

ECxx (...d) =NOEC = approx. 10,000 mg/kg soil dwLOEC = approx. 40,000 mg/kg soil dw

Method: Activated sludge (ca. 13% solids) with test substance were placed over a ca. 4 mm depth of silt loam in a Petri dish.

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The amount of substance tested was mixed with the activated sludge.There were 2 hatchlings per concentration and 5 replicates per concentration.Concentrations: approx. 0, 0.1, 1.0, 4 and 8% (w/w).

Stored at 24oC.GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Aldrich Chemical Co., Milwaukee, WI, USA.

Purity: No data.Remarks: The mortality at LOEC was 80%.Reference: Hartenstein, 1982.

4.6.2 TOXICITY TO TERRESTRIAL PLANTS

(a)Species: Lactuca sativa (Lettuce)End point: Emergence ( x ); Growth ( ); Other ( )Exposure period: 3 daysResults: EC50 /LC50(7d) =

EC50/LC50(14d) =ECxx/LC50(3d) =4.26 (+/-0.20) mmol/lNOEC =LOEC =

Method: Germination tests carried out on agar at 30oC with Lactuca sativa L. cv. Great Lakes as described by Reynolds (1957, 1977) using aqueous solution of Vanillin and inhibitory activity expressed as the millimolar concentration producing 50% reduction in percentagegermination compared with water controls at given temperature.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin. No further data.Remarks: 95% confidence limits.Reference: Reynolds, 1978.

(b)Species: Triticum aestivum L. (Wheat)

Other terrestrial plant: Gossypium hirsutum L. (Cotton)End point: Emergence ( ); Growth ( x ); Other ( )Exposure period: No dataResults: EC50 /LC50(7d) =

EC50/LC50(14d) =ECxx/LCxx(...d) =NOEC =LOEC =

Method: Petri dish bioassayThe potential allelopathic activity of devil's-claw essential oil and a few of the components it contains (incl. Vanillin) on the elongation of cotton and wheat radicles was studied using a Petri dish bioassay.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Sigma Chemical Co., St. Louis, Missouri 63178, USA.

Purity: No data

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Remarks: Vanillin, identified by CGC-MS-DS in the root and pod, was found to be visually observable inhibitory to cotton, but not to wheat at a concentration of 1 mmol in methanol.30 mg Vanillin/dish (2 ml of a 1mmol solution added to the dish) was 11% inhibitory to cotton radicles, but not inhibitory to wheat.

Reference: Riffle et al, 1990.

4.6.3 TOXICITY TO OTHER NON-MAMMALIAN TERRESTRIAL ORGANISMS

Species:End point: Mortality ( ); Reproduction rate ( ); Weight ( ); Other ( )Exposure period:Results: LDxx /LCxx(...d) =

NOEC =LOEC =

Method:GLP: Yes ( ) No ( ) ? ( )Test substance:Remarks: No dataReference:

4.7 BIOLOGICAL EFFECTS MONITORING (INCLUDINGBIOMAGNIFICATION)

Results: Substance:Species or ecosystem studied:Effects monitored:Results:Chemical analysis:

Remarks: No dataReference:

4.8 BIOTRANSFORMATION AND KINETICS IN ENVIRONMENTAL SPECIES

Type: Animal ( ); Aquatic ( ); Plant ( ); Terrestrial ( ); Other ( )Results: No dataRemarks:Reference:


Results: No dataRemarks:Reference:

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5. TOXICITY

5.1 ACUTE TOXICITY

5.1.1 ACUTE ORAL TOXICITY

(a)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Rat, Sprague DawleyValue: 39251 and 39782 mg/kgMethod: OECD TG 401 (1987) and Directive 84/449/EEC, B.1 (1984) "Acute

toxicity (oral)"GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1-1.2. (Lot no. 90-24-701 from Rhône-Poulenc,

France)Purity: 99.9%

Remarks: Single ingastric intubation as a suspension in aqueous solution of 1% (w/v) carboxymethylcellulose at the dose levels 2000, 2510, 3160 and 3980 mg/kg.5 males and 5 females per group.(1) Bliss' method: LD50 = 3978 mg/kg (2484-6368)(2) Litchfield & Wilcoxon's method: LD50 = 3925 mg/kg (2834-5435)Body weight gained similar to control; no macroscopical anomali observed, except some congestive lungs in dead animals.

Reference: Lheritier, 1992.

(b)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Rat, Sprague DawleyValue: 42001, 38002 and 46003 mg/kg bwMethod: OECD TG 401 and Directive 79/831/EEC Annex V, B1.GLP: Yes ( ) No ( ) ? ( x )Test substance: As prescribed by 1.1-1.2. (Batch no. 132 dated 18.09.86, from

EuroVanillin KS, Norway)Purity: 99.8%

Remarks: Dose levels: 2500, 3200, 4000 and 5000 mg/kg.5 males and 5 females per group.95% confidence limits in parenthesis:(1) Males and females combined: LD50 = 4200 mg/kg (3600-5400)(2) Males only: LD50= 3800 mg/kg (2900-5000)(3) Females only: LD50 = 4600 mg/kg (3700-6900)

Reference: Gardner, 1987.

(c)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Rat, Sprague DawleyValue: 3300 mg/kgMethod: Similar to OECD TG 401.GLP: Yes ( ) No ( x ) ? ( )

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Test substance: Vanillin from Monsanto Chemical Company (USA)Remarks: Vanillin in suspension of 10% corn oil.

5 rats per group.Dose levels: 2510, 3160 and 3980 mg/kg.LD50: 3300 mg/kg (3100-3530).At autopsy, lung and liver hypermia and gastrointestinal inflammationin dead animals. Viscera appeared normal in survivors.

Reference: Younger Laboratories Inc., 1976.

(d)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Rat, Osborne MendelValue: 1580 mg/kgMethod: Litchfield & Wilcoxon (1949)GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; commercially available material.Remarks: Vanillin was diluted in propyleneglycol to a 20% (w/v) solution.

10 young adult rats, evenly divided by sex.Were fasted approx. 18 hours prior to treatment.Observation period: 2 weeks.Coma soon after treatment.Death time: 4 hours to 4 days.

Reference: Jenner et al, 1964.

(e)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: RatValue: 2000 mg/kgMethod: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: Single doses of Vanillin as a suspension in corn oil.

95% confidence limits in parenthesis:LD50 = 2000 mg/kg (1600-2500).

Reference: Hake et al, 1963.

(f)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Rat, albinoValue: 3830 mg/kgMethod: Thompson moving average method.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Monsanto Chemical Company, USA.Remarks: Administration as a 20% or 40% suspension in 0,5% solution of

methyl cellulose.5 male rats per group.Dose levels: 2150, 3160, 4640, 6810 and 10,000 mg/kg.Observation period: 7 days.LD50 = 3830 mg/kg (2930-5000).Hemorragic lungs, irritation gastrointestinal, congested kidneys and adrenals in dead animals.No gross pathology in survivors.

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Reference: Hazleton Laboratory, 1955.

(g)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Guinea PigValue: 1400 mg/kg (1310-1500 mg/kg)Method: Litchfield & Wilcoxon (1949).GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; commercially available material.Remarks: Vanillin was diluted in propyleneglycol to a 20% (w/v) solution.

10 guinea pigs, evenly divided by sex.Were fasted approx. 18 hours prior to treatment.Observation period: 2 weeks.Depression within 1 hour.Death time: 1-3 days.

Reference: Jenner et al, 1964.

5.1.2 ACUTE INHALATION TOXICITY

Type:Species/strain:Exposure time:Value: No dataMethod:GLP:Test substance:Remarks:Reference:

5.1.3 ACUTE DERMAL TOXICITY

(a)Type: LD0 ( x ); LD100 ( ); LD50 ( ); LDL0 ( ); Other ( )Species/strain: Rat, Sprague DawleyValue: >= 2000 mg/kgMethod: OECD TG 402 (1987), Directive 84/449/EEC (1984), E.P.A. guideline

no. 798.1100 (1985) and M.A.F.F. guideline no. 4200 (1985).GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1-1.2. (Lot no. 90-24-701 from Rhône-Poulenc,


Remarks: Limit test.5 male and 5 female.Unique dose 2000 mg/kg.A paste of ca. 70% Vanillin in purified water was applied on the shaved skin (10% body area) for 24 hours using semi-occusive patch.Examination after 15 minutes, 1,2 and 4 hours and daily for 14 days.No mortality or pathological clinical sign.No cutaneous lesions.No macroscopic anomali at necropsy.

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Reference: Lheritier, 1991.

(b)Type: LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: RabbitValue: > 5010 mg/kgMethod: OtherGLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Monsanto Chemical Company, USA.Remarks: Applied as a 40% solution suspended in corn oil.

Exposure for 24 hours.1 rabbit per dose level.No mortality after 14 days at 3160 and 5010 mg/kg.Mortality after 3 days at 7940 mg/kg.

Reference: Younger Laboratories Inc, 1976.

5.1.4 ACUTE TOXICITY, OTHER ROUTES OF ADMINISTRATION

(a)Type: LC0 ( ); LC100 ( ); LC50 ( ); LCL0 ( ); Other ( )

LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: RatRoute of administration: i.m. ( ); i.p. ( x ); i.v. ( ); Infusion ( ); s.c. ( ); Other ( )Exposure time: No dataValue: 1160 mg/kgMethod: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: No dataRemarks:Reference: Caujolle et al, 1956.

(b)Type: LC0 ( ); LC100 ( ); LC50 ( ); LCL0 ( ); Other ( )

LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: MouseRoute of administration: i.m. ( ); i.p. ( x ); i.v. ( ); Infusion ( ); s.c. ( ); Other ( )Exposure time: No dataValue: 780 mg/kgMethod: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: No dataRemarks:Reference: Caujolle et al, 1954.

(c)Type: LC0 ( ); LC100 ( ); LC50 ( ); LCL0 ( ); Other ( )

LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: MouseRoute of administration: i.m. ( ); i.p. ( x ); i.v. ( ); Infusion ( ); s.c. ( ); Other ( )Exposure time: No dataValue: 475 mg/kg

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Method: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: No dataRemarks:Reference: Frazer, 1967.

(d)Type: LC0 ( ); LC100 ( ); LC50 ( ); LCL0 ( ); Other ( )

LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Guinea pigRoute of administration: i.m. ( ); i.p. ( x ); i.v. ( ); Infusion ( ); s.c. ( ); Other ( )Exposure time: No dataValue: 1190 mg/kgMethod: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: No dataRemarks:Reference: Caujolle et al, 1954.

(e)Type: LC0 ( ); LC100 ( ); LC50 ( ); LCL0 ( ); Other ( )

LD0 ( ); LD100 ( ); LD50 ( x ); LDL0 ( ); Other ( )Species/strain: Rat, albinoRoute of administration: i.m. ( ); i.p. ( ); i.v. ( ); Infusion ( ); s.c. ( x ); Other ( )Exposure time: Untill deathValue: 2600 mg/kg bw.

Method: Vanillin was dissolved in milk (4%) by slow heating to 90oC, and

cooling to 37oC. Injected subcutaneously into young albino rats.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin.

Purity: high degree of purity, obtained from commercial sources.Remarks: Time till death: 2,5 hours - 4 days.

1000 and 1400 mg/kg had no lethal effect, 1800 mg/kg killed 20% of the rats, and 2200 mg/kg killed 40% of the rats.

Reference: Deichman et al, 1940.

(f)Type: LC0 ( ); LC100 ( ); LC50 ( ); LCL0 ( ); Other ( )

LD0 ( ); LD100 ( ); LD50 ( ); LDL0 ( x ); Other ( )Species/strain: DogRoute of administration: i.m. ( ); i.p. ( ); i.v. ( x ); Infusion ( ); s.c. ( ); Other ( )Exposure time: No dataValue: 1320 mg/kgMethod: Slow i.v. infusionGLP: Yes ( ) No ( x ) ? ( )Test substance: No dataRemarks:Reference: Caujolle et al, 1953.

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5.2 CORROSIVENESS/IRRITATION

5.2.1 SKIN IRRITATION/CORROSION

(a)Species/strain: RabbitResults: Highly corrosive ( ); Corrosive ( ); Highly irritating ( ); Irritating (

); Moderate irritating ( ); Slightly irritating ( ); Not irritating ( x )Classification: Highly corrosive (causes severe burns) ( );

Corrosive (caused burns) ( ); Irritating ( ); Not irritating ( x )Method: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Monsanto Chemical Company, USARemarks: 6 rabbits.

Applicated as a finely ground sample moistened with water.Exposure for 24 h.No irritation.


(b)Species/strain: HumanResults: Highly corrosive ( ); Corrosive ( ); Highly irritating ( ); Irritating (


Corrosive (caused burns) ( ); Irritating ( ); Not irritating ( x )Method: Closed patch test (24-72 hours)GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: No primary irritation when tested in the following closed patch tests:

1)Vanillin concentration: 20%Media: Vaseline Aldum, Unguentum HydrophilicumNumber of subjects: 29 (normal health)Site of application: small of backDuration: 48 hoursResult: Negative in 29 sucjects2)Vanillin concentration: 2%Media: Unguentum Simplex, Unguentum HydrophilicumNumber of subjects: 30 (normal health)Site of application: upper inside of armDuration: 24-72 hoursResult: Negative in 30 subjects3)Vanillin concentration: 0.4 %Media: 99% Ethanol, Non-irritative Cream baseNumber of subjects: 35 (with dermatoses)Site of application: upper inside of armDuration: 24-48 hoursResult: negative in 35 subjects

Reference: Fujii et al, 1972.

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(c)Species/strain: HumanResults: Highly corrosive ( ); Corrosive ( ); Highly irritating ( ); Irritating (); Moderate irritating ( ); Slightly irritating ( ); Not irritating ( x )Classification: Highly corrosive (causes severe burns) ( );

Corrosive (caused burns) ( ); Irritating ( ); Not irritating ( )Method: Closed patch test 48 h.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: Closed patch tests.

Exposure: 48 hours (reading 1 hour later) with pure Vanillin.30 employees (in packaging section, packaging done manually), some ofwhich have/had dermatitis and 15 healthy workers from other units. Negative results only.

Reference: Wang et al, 1987.

(d)Species/strain: Guinea pigResults: Highly corrosive ( ); Corrosive ( ); Highly irritating ( ); Irritating (


Corrosive (caused burns) ( ); Irritating ( ); Not irritating ( )Method: Closed patch test.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: Albino guinea pigs (300-450g), 6-8 weeks old, male and female.

5 pigs:- 1, 2, 5 and 10% preparations of Vanillin in petrolatum.- 0.1 g applied.- Removed after 48 hours, reading 1, 24 and 48 hours later.- Result: negative.10 pigs:- 10% preparation of Vanillin in petrolatum.- 0.1 g applied.- Exposure 24 hours.- Repeated 3 times a week for 2 weeks.- 2 weeks after 2 and 5% preparations and pure Vanillin were appliedand removed 48 hours later.- Reading after 1, 24 and 48 hours.- All negative results.


5.2.2 EYE IRRITATION/CORROSION

(a)Species/strain: RabbitResults: Highly corrosive ( ); Corrosive ( ); Highly irritating ( ); Irritating (

); Moderate irritating ( ); Slightly irritating ( x ); Not irritating ( )Classification: Irritating ( ); Not irritating ( x ); Risk of serious damage to eyes ( )Method: No dataGLP: Yes ( ) No ( x ) ? ( )

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Test substance: Vanillin from Monsanto Chemical Company, USA.Remarks: 6 rabbits.

Application as finely ground powder (dose equivalent to 0.1 ml volume:55 mg sample).Exposure for 24 hours.Irritating score: 18.8/110.Gradually improvement from 48 to 120 hours.All scored zero after 168 hours.


5.3 SKIN SENSITISATION

(a)Type: Buehler testSpecies/strain: Guinea pigResults: Sensitizing ( ); Not sensitizing ( x ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( )Method: Closed patch testGLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: Albino guinea pigs (300-450g), 6-8 weeks old, male and female.

5 pigs:- 1, 2, 5 and 10% preparations of Vanillin in petrolatum.- 0.1 g applied.- Removed after 48 hours, reading 1, 24 and 48 hours later.- Result: negative for all animals.10 pigs:- 10% preparation of Vanillin in petrolatum.- 0.1 g applied.- Exposure 24 hours.- Repeated 3 times a week for 2 weeks.- 2 weeks after 2 and 5% preparations and pure Vanillin were appliedand removed 48 hours later.- Reading after 1, 24 and 48 hours.- Negative results for all animals.


(b)Type: Draize-testSpecies/strain: Guinea pigResults: Negative.

Sensitizing ( ); Not sensitizing ( x ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( x )Method: Similar to Directive 84/449/EEC, B.6.

A dose of 0.05 ml of a 0.1 per cent solution of the compound tested in isotonic saline was injected intradermally on day 0 and further doses of0.1 ml each were injected on 9 alternate days (total dose 0.95 mg).The treated animals and untreated controls were challengedintradermally with 0.05 ml of a 0.1 per cent solution on days 35 and 49.The evaluation criterion was the mean diameter of the papular reactions.

GLP: Yes ( ) No ( x ) ? ( )

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Test substance: Vanillin; no further dataRemarks: 6 to 8 guinea pigs per concentration group. Weight 400-500 g.Reference: Klecak et al, 1977.

(c)Type: Freund's complete adjuvant testSpecies/strain: Guinea pigResults: Sensitizing ( x ); Not sensitizing ( ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( )Method: Doses of 0.05 ml of the undiluted compound mixed with the same

volume of FCA (Freund's complete adjuvant) were injected into the neckon days 0, 2, 4, 7 and 9 (total dose: 250 mg).The control animals were similarly treated with 5x0.05 ml of FCAalone.All the animals were subjects to epicutaneous test on days 21 and 35.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: 6 to 8 guinea pigs per concentration group. Weight: 400-500 g.

No further data on percentage of animals with positive result.Reference: Klecak et al, 1977.

(d)Type: Freund's complete adjuvant test (modified)Species/strain: Guinea pigResults: Weak sensitizing capacity at 10% concentration; mean response: 1.17

(quotient of the sum of all reactions obtained, divided by the totalnumber of animals tested).Sensitizing ( ); Not sensitizing ( ); Ambigous ( x )

Classification: Sensitizing ( ); Not sensitizing ( )Method: See RemarksGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Merck; purified by preparative TLC (thin-layer

chromatography), eluent: chloroform-methanol (100+2) (Haarmann & Reimer, Holzminden, Germany).

Remarks: 10 guinea pigs.Challenge concentration: 10% in acetone.The compounds tested were divided into groups of weak, moderate or strong sensitizing capacity.No further data on percentage of animals with positive result.

Reference: Hausen et al, 1992.

(e)Type: Guinea pig maximization testSpecies/strain: Guinea pig, Dunkin HartleyResults: Sensitizing ( ); Not sensitizing ( x ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( x )Method: OECD TG 406 (1981), Directive 84/499/EEC Annex V, B.6 (1984) and

E.P.A. guideline no 798.4100 (1985).GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1-1.2 (Lot no. 90-24-701 from Rhône-Poulenc,


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Remarks: 40 albino guinea pigs of both sexes; one control group and one treated group.Control group: Induction: vehicle, Challenge: test article (Vanillin crystals).Treated group: Induction and challenge: test article.1) Induction by 3 series of 2 intradermal injections:- FCA- Vanillin 35% in ethanol solution- Vanillin 17.5% in ethanol and FCA2) Topical occlusive application for 48 hours- Vanillin as a 73% paste in ethanol3) Rest period for 11 days4) Topical occlusive application for 24 hours as a 73% paste in ethanol.Signs of irritation were noted during the induction.The test article did not provoke any reaction of cutaneous sensitizationin the animals examined.

Reference: Mercier, 1992.

(f)Type: Guinea pig maximization testSpecies/strain: Guinea pig, HartleyResults: Sensitizing ( x ); Not sensitizing ( ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( )Method: See RemarksGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Allied Corporation.

Purity: >98%Remarks: 15 guinea pigs, 6 controls.

Vehicle: petrolatumConcentrations:Intradermal injection: 50%Topical induction: 50%Challenge (Split adjuvant (Klecak, 1983)): 50%.Induction: 2 intradermal injections, followed by (day 7) closed patch application to id injected skin site for 48 hours.Challenge: Day 21, closed patch application for 24 hours to naive skin site.Positive results in 60% of animals.

Reference: Gad et al, 1986, Gad, 1988.

(g)Type: Guinea pig maximization testSpecies/strain: Guinea pigResults: Sensitizing ( x ); Not sensitizing ( ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( )Method: No dataGLP: Yes ( ) No ( ) ? ( x )Test substance: No dataRemarks: Intradermal injection and/or topical application.

Positive result; 10-50% Vanillin.No further data on percentage of animals with positive result.Moderate sensitizer.

Reference: Ishihara et al, 1986.

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(h)Type: Guinea pig maximization testSpecies/strain: Guinea pigResults: Sensitizing ( x ); Not sensitizing ( ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( )Method: Induction:

Day 0: Intradermal injection of 0.1 ml of a 5% solution of thecompound, of 0.1 ml of a 5% emulsion of the compound in FCA(Freund's complete adjuvant) and of 0.1 ml of FCA alone. Each injectionwas given twice.Day 8: Application, for 2 days under occlusive bandage to a clippedskin area of the neck, of 250 mg of the compound dissolved inpetrolatum at a concentration of 25% (wich always causes mild tomoderate skin irritation under occlusion). Total dose: 20 mgintradermally plus 250 mg epicutaneously.Challenge:Day 21: Occlusive patch test at a subirritant concentration (unspecified)in petrolatum, applied to the flank for 24 hours. Reading 24 and 48hours after removing the patch.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataRemarks: 6 to 8 guinea pigs per concentration group.

No further data on percentage of animals with positive result.Reference: Klecak et al, 1977.

(i)Type: Open epicutaneous testSpecies/strain: Guinea pigResults: Not sensitizing. Undiluted Vanillin: positive.

Sensitizing ( ); Not sensitizing ( ); Ambigous ( x )Classification: Sensitizing ( ); Not sensitizing ( )Method: Induction:

Application of 0.1 ml undiluted compound and diluted solutions toshaved skin, repeated daily for 21 days, using the same skin site.Applications left uncovered. Reading 24 hours after each application.Minimum irritation concentration was determined.Minimum irritation concentration: 30% after 1 application, 3% after 21 applications.The same guinea pigs (+6-8 untreated controls), were tested on days 21 and 35 on contralateral flank, at the minimal irritating concentration and at some lower non-irritant concentrations (unspecified).Reading after 24, 48 and 72 hours.The minimal sensitizing concentration necessary to induce contact hypersensitivity was determined. A concentration was concidered allergenic when at least 2 out of 8 animals showed positive reactionswith non-irritant concentrations used for challenge, based on practical experience.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin: no further dataRemarks: Test concentrations: Undiluted, dissolved at concentrations of 30, 10, 3,

1, 0.3, 0.1 and 0.03 %.6 to 8 guinea pigs per concentration group.

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Reference: Klecak et al, 1977.

(j)Type: Other: Maximization testSpecies/strain: HumanResults: Sensitizing ( ); Not sensitizing ( x ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( )Method: Kligman Maximization TestGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin; no selections for pure grades, materials were picked as actually

used.Remarks: 25 healthy adults.

Patch test: 1.0 ml of 5% aqueous sodium lauryl sulfate solution for 24 hours to produce a moderate inflammatory reaction and render the skin more permeable to the test agent (1).Same site: 48 hour occlusive patch applied with test material (2).(1) and (2) are alternated for a total 5 exposures of each, period of 15 days, 10 days rest period.Challenge.A new site: 10% solution was applied for 1 hour. Washed off. Test material was applied to this new pretreated area; occlusive patch for 48hours.Test concentration: 2%.Test area examined immediately and at 2 successive days.No sensitizing reactions were induced in groups of 25 volunteers.

Reference: Greif, 1967.

(k)Type: Mouse ear swelling testSpecies/strain: Mouse, CF-1Results: No animal sensitized.

Sensitizing ( ); Not sensitizing ( x ); Ambigous ( )Classification: Sensitizing ( ); Not sensitizing ( x )Method: Alternative OECD methodGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Allied Corp.

Purity: > 98%Remarks: 10-15 female mice, 5-10 in control group.

Induction: Topical application at days 0, 1, 2, 3 and 4 to abdominal skinprepared by FCA intradermal injection.Challenge: Day 10, topical application of test substance to one ear, of vehicle to the other.Ear thickness measurement after 24 and 48 hours.Vanillin 50% in ethanol 70%.

Reference: Gad et al, 1986, Gad, 1988.

5.4 REPEATED DOSE TOXICITY

(a)Species/strain: Rat, Osborne-MendelSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )

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Route of administration: Oral feedExposure period: 16 weeksFrequency of treatment: DailyPostexposure observ. period: NoneDose: 10,000 ppmControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( x ); Historical ( )NOEL: >= 10,000 ppmLOEL:Results: No effect on growth or haematology.

No macroscopic or microscopic changes in the tissues (incl. testes).Method: 5 male and 5 female rats (test and control groups).

Vanillin was mixed in the diet. Fresh diets were made and distributed weekly. The concentration was adjusted so that all rats received aconstant volume of 1 ml of solution/kg daily.The rat's weight, food intake and general condition were recorded everyweek.Haematological examinations were made at termination of the study.These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.At the termination of the experiments the rats were sacrificed and exsaguinated. The tissues of all the rats were examined macroscopicallyat the time of sacrifice.The viscera were removed and the liver, kidneys, spleen, heart and testeswere weighed.These organs, the remaining abdominal and thoracic viscera, and onehind leg, for bone, bone marrow, and muscle, were preserved in 10%buffered formalin-saline solution for histopathological examination.For routine histopathology, sections were embedded in paraffin wax andstained with haematoxylin and eosin.Detailed microscopic examinations in the subacute studies weregenerally done on 6 or 8 rats, evenly devided by sex, from the high dosegroup and the control group.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; commercially available.Reference: Hagan et al, 1967.

(b)Species/strain: Rat, Osborne-MendelSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )Route of administration: Oral feedExposure period: 27-28 weeksFrequency of treatment: DailyPostexposure observ. period: NoneDose: 1000 ppmControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( x ); Historical ( )NOEL: >= 1000 ppmLOEL:Results: No effect on growth or haematology.


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Vanillin was mixed in the diet. Fresh diets were made and distributed weekly. The concentration was adjusted so that all rats received aconstant volume of 1 ml of solution/kg daily.The rat's weight, food intake and general condition were recorded everyweek.Haematological examinations were made after 3 months and attermination of the study.These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.At the termination of the experiments the rats were sacrificed andexsaguinated. The tissues of all the rats were examined macroscopicallyat the time of sacrifice.The viscera were removed and the liver, kidneys, spleen, heart and testeswere weighed.These organs, the remaining abdominal and thoracic viscera, and onehind leg, for bone, bone marrow, and muscle, were preserved in 10%buffered formalin-saline solution for histopathological examination.For routine histopathology, sections were embedded in paraffin wax andstained with haematoxylin and eosin.Detailed microscopic examinations in the subacute studies weregenerally done on 6 or 8 rats, evenly devided by sex, from the high dosegroup and the control group.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; commercially availableReference: Hagan et al, 1967.

(c)Species/strain: Rat, Osborne-MendelSex: Female ( ); Male ( x ); Male/Female ( ); No data ( )Route of administration: Oral feedExposure period: 1 yearFrequency of treatment: DailyPostexposure observ. period: NoneDose: 20,000 and 50,000 ppmControl group: Yes ( x ); No ( ); No data ( )


No macroscopic or microscopic changes in the tissues (incl. testes).Method: 5 male rats (test and control groups).

Vanillin was mixed in the diet. Fresh diets were made and distributed weekly. The concentration was adjusted so that all rats received aconstant volume of 1 ml of solution/kg daily.3% (w/w) corn oil added to control or test diets as a binder to reduce evaporation of the flavouring.The rat's weight, food intake and general condition were recorded everyweek.Haematological examinations were made after 3, 6, and 12 months.These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.

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At the termination of the experiments the rats were sacrificed andexsaguinated. The tissues of all the rats were examined macroscopicallyat the time of sacrifice.The viscera were removed and the liver, kidneys, spleen, heart and testeswere weighed.These organs, the remaining abdominal and thoracic viscera, and onehind leg, for bone, bone marrow, and muscle, were preserved in 10%buffered formalin-saline solution for histopathological examination.For routine histopathology, sections were embedded in paraffin wax andstained with haematoxylin and eosin.Detailed microscopic examinations in the subacute studies weregenerally done on 6 or 8 rats, evenly devided by sex, from the high dosegroup and the control group.


(d)Species/strain: Rat, Osborne-MendelSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )Route of administration: Oral feedExposure period: 2 yearsFrequency of treatment: DailyPostexposure observ. period: NoneDose: 5000, 10,000 and 20,000 ppmControl group: Yes ( x ); No ( ); No data ( )



Vanillin was mixed in the diet. Fresh diets were made and distributed weekly. The concentration was adjusted so that all rats received aconstant volume of 1 ml of solution/kg daily.3% (w/w) propylene glycol added to control or test diets as a binder to reduce evaporation of the flavour.The rat's weight, food intake and general condition were recorded everyweek.Haematological examinations were made at 3, 6, 12 and 22 months.These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.At the termination of the experiments the rats were sacrificed andexsaguinated. The tissues of all the rats were examined macroscopicallyat the time of sacrifice.The viscera were removed and the liver, kidneys, spleen, heart and testeswere weighed.These organs, the remaining abdominal and thoracic viscera, and onehind leg, for bone, bone marrow, and muscle, were preserved in 10%buffered formalin-saline solution for histopathological examination.For routine histopathology, sections were embedded in paraffin wax andstained with haematoxylin and eosin.

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Detailed microscopic examinations in the subacute studies weregenerally done on 6 or 8 rats, evenly devided by sex, from the high dosegroup and the control group.


(e)Species/strain: RatSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )Route of administration: Oral feedExposure period: 91 daysFrequency of treatment: No dataPostexposure observ. period: No dataDose: 3000, 10,000 and 50,000 ppm (ca. 150, 500 and 2500 mg/kg/day)Control group: Yes ( ); No ( ); No data ( x )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL: >= 3000 ppmLOEL: >= 10,000 ppmResults: When judged by appearance, behaviour, growth, mortality, final body

and organ weights, terminal hematological examination, and histologigalstudies, no adverse effects were detected at 3000 ppm.Mild adverse effects at 10 000 ppm, Growth depression andenlargement of liver, kidney and spleen at 50,000 ppm.

Method: 10 male and 10 female rats. 4 to 6 weeks of age. No further data.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; no further dataReference: Hake et al, 1963.

(f)Species/strain: RatSex: Female ( ); Male ( x ); Male/Female ( ); No data ( )Route of administration: Oral feedExposure period: 26 weeksFrequency of treatment:Postexposure observ. period: NoneDose: 1000, 5000 and 10,000 ppm (or 0.1%, 0.5% and 1.0%)Control group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL: >= 10,000 ppmLOEL:Results: No significant difference in body weight gain.

Autopsies and microscopic examinations of tissues revealed nopathology.

Method: 10 male rats per group. No further data.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Monsanto Chemical Company, USA.Reference: Hazleton Laboratory, 1955.

(g)Species/strain: RatSex: Female ( ); Male ( ); Male/Female ( ); No data ( x )

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Route of administration: GavageExposure period: 14 weeksFrequency of treatment: Twice a weekPostexposure observ. period: No dataDose: 300 mg/kgControl group: Yes ( ); No ( ); No data ( x )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL: >= 300 mg/kgLOEL:Results: No adverse effects.

Appearance, behaviour and gain in weight were normal.Method: 12 young albino rats.

4% solution in olive oil. Blunt hypodermic needle introduced into the esophagus.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; high degree of purity from commercial sources.Reference: Deichman et al, 1940.

(h)Species/strain: RatSex: Female ( ); Male ( ); Male/Female ( ); No data ( x )Route of administration: Oral feedExposure period: 18 weeksFrequency of treatment: DailyPostexposure observ. period: No dataDose: approx. 20 mg/kg/dayControl group: Yes ( ); No ( ); No data ( x )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL: >= 20 mg/kg bw/dayLOEL:Results: No adverse effects.

Appearance, behaviour and gain in weight were normal.Method: 16 young albino rats.

4% solution in milk, in a normal diet.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; high degree of purity from commercial sources.Reference: Deichman et al, 1940.

(i)Species/strain: RatSex: Female ( ); Male ( ); Male/Female ( ); No data ( x )Route of administration: Oral feedExposure period: 10 weeksFrequency of treatment: DailyPostexposure observ. period: No dataDose: 64 mg/kg/dayControl group: Yes ( ); No ( ); No data ( x )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL:LOEL: >= 64 mg/kg bw/dayResults: Appeared normal and lively, but the rate of increase in weight was

retarded.

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Histopathological changes in the myocardium, liver, kidney, lung, spleenand stomach.

Method: 16 young albino rats.4% solution in milk, in a normal diet.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; high degree of purity from commercial sources.Reference: Deichman et al, 1940.

(j)Species/strain: DogSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )Route of administration: Other: capsuleExposure period: 26 weeks and 4 daysFrequency of treatment: 5 days a weekPostexposure observ. period: NoneDose: 0, 25 and 100 mg/kg.Control group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL: >= 100 mg/kgLOEL:Results: 1 male and 1 female dog per dose.

Normal behaviour and body weight gains.Hematological or biochemical values and urine analysis for all treatedanimals were within normal limits and comparable to the control values.Gross autopsies and microscopic examinations of tissues revealed no pathology.

Method: No dataGLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Monsanto Chemical Company, USA.Reference: Hazleton Laboratory, 1955.

5.5 GENETIC TOXICITY IN VITRO

A. Bacterial test

(a)Type: Ames test (Salmonella/mammalian-microsome reverse mutation assay)System for testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538Concentration: 100, 333, 667, 1000, 3330 and 5000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Vanillin did not cause a positive increase in the number of histidine

revertants per plate of any of the tested strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver(S9).

Cytotoxicity conc.: With metabolic activation: > 5000 ug/plateWithout metabolic activation: > 5000 ug/plate

Precipitation conc.:Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( x )

Without metabolic activation: + ( ) ? ( ) - ( x )Method: OECD TG 471 "Genetic Toxicology: Salmonella thyphimurium Reverse

Mutation Assay".

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GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescibed by 1.1-1.2 (Sample from Rhône Poulenc)

Purity: no data (most probably > 99.6%)Remarks:Reference: Lawlor, 1991.

(b)Type: Ames test (Bacterial reverse gene mutation assay)System for testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538Concentration: 50, 150, 500, 1500 and 5000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: No substantial increases in revertant colony numbers of any of the five

tester strains were observed following treatment with Vanillin at anydose level, either in the presence or absence of metabolic activation (S-9mix).It was concluded that no evidence of mutagenic potential of Vanillin wasobtained in this bacterial test system at the dose levels used.



Without metabolic activation: + ( ) ? ( ) - ( x )Method: Bacterial reverse gene mutation assay (Ames test)GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1-1.2 (Batch no. 132 dated 18.09.86 from

EuroVanillin KS, Norway)Purity: 99.8%

Remarks:Reference: Jones et al, 1986.

(c)Type: Ames test (Salmonella/mammalian-microsome reverse mutation assay)System for testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537Concentration: 100, 333, 1000, 3333 and 10,000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative

Cytotoxicity conc.: With metabolic activation: > 10,000 ug/plateWithout metabolic activation: > 10,000 ug/plate


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Salmonella Mutagenicity Test.

With and without Aroclor 1254-induced rat and hamster metabolic activation systems.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Aldrich

Purity: 99%Remarks: Cytotoxicity at 10,000 ug/plate.Reference: Mortelmans et al. 1986.

(d)Type: Ames test

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System for testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537Concentration: 8, 40, 200 and 1000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative

Cytotoxicity conc.: With metabolic activation:Without metabolic activation:

Precipitation conc.:Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( )

Without metabolic activation: + ( ) ? ( ) - ( )Method: OECD TG 471 "Genetic Toxicology: Salmonella thyphimurium Reverse

Mutation Assay".GLP: Yes ( ) No ( x ) ? ( )Test substance: As prescribed by 1.1-1.2

Purity: no exact dataRemarks:Reference: Marzin, 1979a.

(e)Type: Ames testSystem for testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538Concentration: 0,5, 5, 50, 500 and 5000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative at 0,5, 5, 50 and 500 ug/plate.

At 5000 ug/plate; toxicity as evidenced by a thinning of the backgroundlawn.Vanillin did not show any mutagenicity in the Ames assay.



Without metabolic activation: + ( ) ? ( ) - ( x )Method: Salmonella thyphimurium Assay.GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Schuchardt, Munich.

Purity: no dataRemarks: Tested in quadruplicate at five concentrations on each of the 5 bacterial

strains, both in presence and absence of the S9-mix.Reference: Pool et al, 1982.

(f)Type: Ames testSystem for testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537Concentration: 3 umol/plate (or 456 ug/plate)Metabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative.

Not mutagenic.Cytotoxicity conc.: With metabolic activation: > 456 ug/plate

Without metabolic activation: > 456 ug/platePrecipitation conc.: Did not precipitate at 456 ug/plate.Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( x )

Without metabolic activation: + ( ) ? ( ) - ( x )Method: Spot test

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GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin; commercially available, checked for purity using thin-layer

chromatography, gas chromatography and NMR.Remarks: Metabolic activation: S-9, Aroclor 1254.Reference: Florin et al. 1980.

(g)Type: Ames test (Salmonella/microsome test - reverse mutation assay)System for testing: Salmonella typhimurium TA92, TA94, TA98, TA100, TA1535,

TA1537Concentration: 6 concentrations up to 10,000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative.

No significant increase in the numbers of revertant colonies weredetected in any of the strains at the maximum dose (10,000 ug/plate).

Cytotoxicity conc.: With metabolic activation: > 10,000 ug/plateWithout metabolic activation: > 10,000 ug/plate


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Salmonella/microsome test - reverse mutation assay. Ames test.

Liver microsome fraction (S-9) was prepared from the liver of Fischer rats.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from the Japan Food Additives Association, Tokyo.

Purity: checked, no further dataRemarks:Reference: Ishidate et al, 1984.

(h)Type: Ames test (bacterial mutation test in the Salmonella/microsome system)System for testing: Salmonella typhimurium TA98, TA100Concentration: 0.05 to 1000 ug/plateMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative.

Did not induce a number of revertants that was over half the number ofspontaneous revertants of TA98 and TA100 at the indicated doseranges, either with or without S9 mix.



Without metabolic activation: + ( ) ? ( ) - ( x )Method: Bacterial mutation test in the Salmonella/microsome system.

Rat liver microsome (S-9) was prepared from Sprague-Dawley rats treated with Aroclor 1254 by the metod of Ames.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Nakarai Pharmaceutical Co. Ltd., Japan.

Purity: 90-95%Remarks: Metabolic activation: Rat-liver microsome (S 9) from Sprague-Dawley

rats treated with Aroclor 1254.Reference: Kasamaki et al, 1982.

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(i)Type: Salmonella typhimurium reverse mutation assay (Modified Ames test)System for testing: Salmonella typhimurium TA102, TA104Concentration: 0, 2, 4 6, 8, and 10 umol/plate (10 uM = 1520 ug)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

Vanillin was a powerful inhibitor of the spontaneous mutagenicity inboth TA104 (ca. 50% decrease) and TA102 (ca. 40% decrease).

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: > 10 umol/plate


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Inhibition of the spontaneous mutagenicityGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Aldrich.

Purity: no dataRemarks:Reference: De Flora et al, 1994.

(j)Type: Salmonella typhimurium reverse mutation assay (modified Ames test)System for testing: Salmonella typhimurium TA98, TA100, TA1535Concentration: No dataMetabolic activation: With ( x ); Without ( ); With and without ( ); No data ( )Results: Negative.

Vanillin was non-mutagenic.Cytotoxicity conc.: With metabolic activation:

Without metabolic activation:Precipitation conc.:Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( x )

Without metabolic activation: + ( ) ? ( ) - ( x )Method: Salmonella typhimurium reverse mutation assay .

S9 fraction from the livers of Aroclor-induced Wistar rats and S9 activation mix were prepared according to the method of McCann et al (1975).

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Sisco Laboratories, Bombay

Purity: 90%Remarks: Metabolic activation: S9 from livers of Aroclor-induced Wistar rats.Reference: Nagabhushan et al, 1985.

(k)Type: Bacterial reverse mutation assaySystem for testing: Escherichia coli WP2s (uvrA, trpE), Salmonella typhimurium TA98

(uvrB, hisD)Concentration: 50, 100 and 150 ug/plateMetabolic activation: With ( x ); Without ( ); With and without ( ); No data ( )Results: #Antimutagenic effects of Vanillin post-treatment on mutagenesis

induced in E. coli:- by 4-Nitroquinoline 1-oxide (at 2 ug/ml for 15 minutes):

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Strong suppressing activity, marked increase in the survival of treated cells.- by Furylfuramide (at 0.4 ug/ml for 60 minutes):Strong suppressive activity.- by Captan (at 2 ug/ml for 15 minutes):Strong suppressive activity.- by Methylglyoxal (at 100 ug/ml for 60 minutes):Strong suppressive activity.#In Salmonella typh. TA98 Vanillin was not effective against mutationsprovoked by:- 3-Amino-1-methyl-5H-pyrido(3-4-b) indole- 2-Amino-3-methylimidazo (4-5-f) quinolinein Salmonella.

Cytotoxicity conc.: With metabolic activation: >150 ug/plateWithout metabolic activation: > 150 ug/plate


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Assay for antimutagenic effectsGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo Japan

Purity: no dataRemarks: Metabolic activation: S-9.Reference: Ohta et al, 1986.

(l)Type: DNA damage and repair assaySystem for testing: Escherichia coli K-12 and PJ5Concentration: 0,5, 1, 3, 5 and 10 mmol/l (1520 mg/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

Vanillin has an effect on the adaptive and SOS responses, as well asmutagenesis, induced in E.coli by N-methyl-N-nitroso urea (MNU) andUV-irradiation.Vanillin, to some extent, suppressed MNU-induced mutagenesis in bothDNA repair-proficient strain (AB1157) and the ada-5 mutant (PJ5) ofE. coli K-12. However, when E.coli K-12 AB1157 cells were treatedwith MNU in a buffer followed by incubation in a growth mediumcontaining vanillin, MNU-induced mutagenesis was not suppressed.It is therefore suggested that Vanillin might suppress the methyl-actionof DNA by MNU.Concerning UV-induced mutagenesis, vanillin suppressed mutagenesisunder the assay conditions in E.coli K-12 AB1157. Vanillin alone areincapable of inducing any revertants over the background level under thepresent assay conditions in either DNA repair-proficient (AB1157) orthe ada-5 mutant (PJ5).

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: > 1520 mg/l


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Effects on MNU- and UV-induced mutagenesis.

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The effect on MNU-induced mutagenesis were examined by measuringrevertant cells formed after incubation of E.coli K-12 tester strains in thepresence of Vanillin.For the effect on UV-induced mutagenesis, revertant cells weremeasured after postincubation of the UV-preirradiated E. coli K-12AB1157 cells in a growth medium containing various concentrations ofVanillin.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Wako Pure Chemicals Industries Ltd., Osaka Japan.

Purity: no dataRemarks:Reference: Takahashi et al, 1990.

(m)Type: DNA damage and repair assaySystem for testing: Escherichia coli K-12 (several strains), plasmids.Concentration: 600 ug/l (UV-survival)

500 ug/l (Plasmid recombination)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: UV-irradiated uvrA umuC cells showed higher survival when plated on

medium containing vanillin rather than medium without vanillin.Antimutagenic effects of Vanillin on UV killing of umuC mutantstrains of E.coli.A significantly higher frequency of plasmid recombination was observedwhen vanillin was present in the culture medium.The study suggest that the antimutagenic effect of vanillin is a result ofenhancement of recA-dependent, error free, pathway of post replicationrepair.



Without metabolic activation: + ( ) ? ( ) - ( )Method: Antimutagenic effect of vanillin on UV-irradiated E.coli.

Antimutagenic activity (UV-survival), post replication repair, plasmid recombination.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo Japan.

Purity: no dataRemarks:Reference: Ohta et al, 1988.

(n)Type: Escherichia coli reverse mutation assaySystem for testing: Escherichia coli WP2s (uvrA, trpE)Concentration: 0, 20 and 30 umol/plate (30 uM = 4560 ug)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Bio-antimutagenic effects of Vanillin on mutagenesis induced by 4-

Nitroquinoline 1-oxide, at 2 ug/ml for 15 minutes: Strong suppressive activities.

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: > 30 umol/plate

Precipitation conc.:

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Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( )Without metabolic activation: + ( ) ? ( ) - ( x )

Method: E. coli trp+ reverse mutation assay.Antimutagenic effects

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo.

Purity: no dataRemarks:Reference: Watanabe et al, 1988.

(o)Type: Escherichia coli reverse mutation assaySystem for testing: Escherichia coli PQ37Concentration: 15, 50, 150, 500 and 1500 ugMetabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Vanillin itself did not cause SOS induction.

Simultaneous treatment with UV irradiation, or with 4-Nitroquinoline- oxide or with N-methyl-N'-nitro-N-nitroso-guanidine increased the mutagenicity.



Without metabolic activation: + ( ) ? ( ) - ( )Method: SOS chromotestGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Kisida Chemical Co., Japan.

Purity: no dataRemarks:Reference: Sato et al, 1991.

(p)Type: Escherichia coli reverse mutation assaySystem for testing: Escherichia coli PQ37Concentration: No data reportedMetabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Ambigous.

Vanillin nitrosated with Sodium nitrite showed strong genotoxicity:SOS-inducing factor = 0,21/nmol, calculated on the basis of 2 to 4duplicate determinations of SOS-inducing factor at variousconcentrations (not reported).



Without metabolic activation: + ( ) ? ( ) - ( )Method: SOS chromotestGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin after nitrosation with Sodium nitrite.

Purity: no dataRemarks:Reference: Oshima et al, 1989.

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(q)Type: Mitotic recombination in Saccharomyces cerevisiae (Mitotic gene

conversion assay)System for testing: Saccharomyces cerevisiae strain D7Concentration: 10 mg/ml (65.8 mM/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

No induction of gene conversion at acidic pH levels.No significantly difference in gene conversion frequency at pH7 and at pH10.



Without metabolic activation: + ( ) ? ( ) - ( )Method: Mitotic recombination in Saccharomyces cerevisiae (Mitotic gene

conversion assay)GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Sigma Chemical Co., St. Louis, MO USA.

Purity: no dataRemarks:Reference: Rosin, 1984.

B. Non-bacterial in vitro test

(a)Type: Cytogenetic assaySystem for testing: Human lymphocytesConcentration: 0, 1, 2 and 4 mmol/l (4mM = 612 mg/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Ambigous.

2 experiments were made with lymphocytes from 2 different donors.One experiment showed a negative result (no increase in SCE above thatof the solvent control), while in the other a slight increase in the numberof aberrations/a slight increase in the chromosome gap was seen withincreasing concentration of Vanillin.However, only the highest concentration (4 mmol/l) showed astatistically significance effect with gaps included (p<0,01).

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: > 4 mmol/lAt the concentration of 6 mmol vanillin it was not possible to obtain enough metaphases to do an evaluation


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Similar to OECD TG 473. (Chromosome aberration)GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin; commercially available

Purity: 99.6% (estimated by gas chromatography)Remarks: Such concentration is neighter physiologic nor encountered at exposure.

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Gaps are not included in the evaluation of chromosome aberration tests according to OECD Guidelines for Testing of Chemicals..

Reference: Jansson, et al. 1987.

(b)Type: Cytogenetic assaySystem for testing: BALB/c mouse 3T3 fibroblastsConcentration: 2 to 8 mmol/l (8mM = 1224 mg/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Relative cytotoxicity NR50 (midpoint cytotoxicity) = 8 mmol/l.

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: 8 mmol/l


Without metabolic activation: + ( ) ? ( ) - ( )Method: Neutral red (NR) assay (quantification of the number of viable,

uninjured cells after their incubation with test agents, based on theuptake and lysosomal accumulation of the supravital dye NR).

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from J.T. Baker, Danvers, MA, USA (dissolved in 95%

ethanol).Purity: no data

Remarks: Microscopic examination: Vanillin, at slight to moderately toxic concentration (2-6 mmol/l), induced multinucleation in the 3T3 fibroblasts, giving rise to two or multiple nuclei.This concentration is neither physiologic nor related to exposure.

Reference: Babich et al, 1993.

(c)Type: DNA damage and repair assaySystem for testing: Chinese Hamster ovary (CHO K-1) cellsConcentration: 10, 33 and 100 umol/l (100uM = 15200 ug/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

Vanillin did not induce chromosome aberrations.Chromosome aberrations induced by UV-light or X-rays weresuppressed by the post-treatment with Vanillin.UV- or X-ray irradiated surviving cells increased in the presence of Vanillin.



Without metabolic activation: + ( ) ? ( ) - ( x )Method: Chromosome aberrations induced by UV-light or X-raysGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo, Japan (dissolved in dimethyl

sulfoxide just before each treatment)Purity: no data

Remarks:Reference: Sasaki et al, 1990a.

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(d)Type: Mammalian cell gene mutation assaySystem for testing: Chinese Hamster ovary (CHO) cellsConcentration: 250 to 1500 ug/ml (-S9 mix), 183 to 2440 ug/ml (+S9 mix)Metabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Vanillin was concidered negative for inducing chromosomal aberrations

in CHO cells under both non-activation and activation, except at thehighest dose level analyzed (2440 ug/ml) with metabolic activation (seecomments below).

Cytotoxicity conc.: With metabolic activation: Rangefinding assay: Complete cytotoxicity at5090 ug/ml, no cell cycling delay. 2440 ug/ml caused severecytotoxicity.*No increase in chromosomal aberrations was observed atthe closely spaced subsequent dose level of 1830 ug/ml and lower doses.

Without metabolic activation: Rangefinding assay: Completecytotoxicity at 1700 and 5090 ug/ml, severe cell cycle delay at 509ug/ml.


Without metabolic activation: + ( ) ? ( ) - ( x )Method: OECD TG 473 "Genetic Toxicology: In vitro Mammalian Cytogenetic

Test".GLP: Yes ( x ) No ( ) ? ( )Test substance: As prescribed by 1.1-1.2. (Lot no. 8932101 from Rhône-Poulenc)

Purity: 99.9%Remarks: *) Aberrations observed at 2440 ug/ml were mostly simple chromosome

breaks localized mainly at a single site. The biological significance ofthis response at an extremely toxic dose level is highly disputable.

Reference: Murli, 1991.

(e)Type: Mammalian cell gene mutation assaySystem for testing: Chinese Hamster fibroblast cell line (CHL, from a lung of a newborn

female)Concentration: 3 different doses up to 1,0 mg/ml for 24 hours and 48 hoursMetabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: >1,0 mg/ml


Without metabolic activation: + ( ) ? ( ) - ( x )Method: Chromosomal aberration test.GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from the Japan Food Additives Association, Tokyo.

Purity: checked, no further dataRemarks:Reference: Ishidate et al, 1984.

(f)Type: Mammalian cell gene mutation assay

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System for testing: Chinese Hamster V79 cellsConcentration: 0, 10, 33 and 100 umol/l (100 uM = 15,200 ug/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: >100 umol/l


Without metabolic activation: + ( ) ? ( ) - ( x )Method: 6-TG (thioguanine) -resistant mutation testGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo, Japan.

Purity: no dataRemarks: The frequencies of 6-TG-resistant mutations induced by UV or X-rays

were decreased by treatment with Vanillin during the expression time. These decreases were depentent on the concentrations of Vanillin.Vanillin was not mutagenic to V79 cells and obvious increases or decreases in the frequency of spontaneous mutations were not observed.Antimutagenic effects: The number of surviving cells after irradiation increased in the presence of Vanillin. The frequencies of 6-TG-resistantmutants induced by UV or X-rays were decreased after 5-, 6-, 7- and 8-day expression times. No prolongation of growth rate was observed.Hence, the observed decreased mutation frequency was not due to thecytotoxicity of Vanillin or a delay in mutation fixation.

Reference: Imanishi et al, 1990.

(g)Type: Mammalian cell gene mutation assaySystem for testing: Chinese Hamster B241 cellsConcentration: 0.005, 0.02 and 0.04 umol/lMetabolic activation: With ( ); Without ( ); With and without ( x ); No data ( )Results: Negative.

No significant increase in structural and numerical chromosome aberrations, compared to control cells untreated or treated with dimethylsulfoxide alone.

Cytotoxicity conc.: With metabolic activation: > 0.04 umol/lWithout metabolic activation: > 0.04 umol/l


Without metabolic activation: + ( ) ? ( ) - ( x )Method: With another cell line, similar to OECD TG 476.

Chromosome aberrations.GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Nakarai Pharmaceutical Co. Ltd., Japan.

Purity: 90-95%Remarks: Metabolic activation: S9 mix.Reference: Kasamaki et al, 1982, Kasamaki et al, 1985.

(h)Type: Sister chromatid exchange assaySystem for testing: Chinese Hamster ovary (CHO) cellsConcentration: 10, 33, 100 and 333 umol/l (333 uM = 50616 ug/l)

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Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Negative.

Vanillin induced neither SCEs nor chromosome aberrations by itself.However, an obvious increase in frequencies of SCEs was observedwhen mytomycine C (MMC)-pretreated cells were cultured in thepresence of Vanillin.SCEs-enhancing effects of Vanillin were also observed when inducedby:- EMS (ethyl methanesulphonate)- EMNG (N-ethyl-N'-nitro-N-nitrosoguanidine)- ENU (N-ethyl-N-nitrosourea)- MNU (N-methyl-N-nitrosourea)On the other hand, MMS (methyl methanesulphonate) or MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-induced SCEs were not influencedat all by Vanillin.

Cytotoxicity conc.: With metabolic activation:Without metabolic activation: >= 333 umol/l (for MMS- or

MNNG-pretreated cells).Precipitation conc.:Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( )

Without metabolic activation: + ( ) ? ( ) - ( )Method: In vitro Sister Chromatid Exchange Assay in Mammalian Cells.GLP: Yes ( ) No ( ) ? ( x )Test substance: Other TS: from Tokyo Kasei Kogyo, Tokyo Japan.

Purity: no dataRemarks: SCEs-enhancing effects seemed to be depentent on the quality of lesions

in DNA.Reference: Sasaki et al, 1987a, Sasaki et al, 1987b.

(i)Type: Sister chromatid exchange assaySystem for testing: Human lymphocytesConcentration: 0.75 and 1.0 mmol/l (1mM = 152 mg/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Vanillin was a potent inducer of SCE, significant at 0.75 and 1.0

mmol/l.Cytotoxicity conc.: With metabolic activation:

Without metabolic activation:Precipitation conc.:Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( )

Without metabolic activation: + ( x ) ? ( ) - ( )Method: Similar to OECD TG 479GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin; commercially available

Purity: 99.6% (estimated by gas chromatography)Remarks: Nothing is said about the toxicity of Vanillin as such huge

concentrations as 1 mM, although Vanillin was studied as a componentof cigarette smoke and present as such at lower concentration.

Reference: Jansson et al, 1986.

(j)Type: Sister chromatid exchange assaySystem for testing: Human lymphocytes

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Concentration: 0, 1and 2 mmol/l (2 mM = 306 mg/l)Metabolic activation: With ( ); Without ( x ); With and without ( ); No data ( )Results: Positive.

Significant (p<0.001) at 1 and 2 mmol/l.Cytotoxicity conc.: With metabolic activation:

Without metabolic activation:Precipitation conc.:Genotoxic effects: With metabolic activation: + ( ) ? ( ) - ( )

Without metabolic activation: + ( ) ? ( ) - ( )Method: Similar to OECD TG 479GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin; commercially available

Purity: 99.6% (estimated by gas chromatography)).Remarks: This study repeats the results from (Jansson et al, 1986) (which tested a

longer list of compounds) about the induction of SCE by Vanillin.Comments in article: The results are probably suggesting a low abilityof Vanillin to induce chromosome aberrations in human lymphocytes.

Reference: Jansson et al, 1987.

5.6 GENETIC TOXICITY IN VIVO

(a)Type: Micronucleus assaySpecies/strain: Mouse, OF1Sex: Female ( x ); Male ( ); Male/Female ( ); No data ( )Route of administration: Gavage (orally)Exposure period: 6 and 30 hours (before sampling of bone-marrow)Doses: 500 and 1000 mg/kg, twice each.Results: Negative.

No statistically significant increase in the frequency of micronuclei in erythrocytes when compared to the control.Lowest dose producing toxicity: 2000 mg/kg (2 lethalities out of 3).

Effect on mitoticindex or P/N ratio:Genotoxic effects: + ( ) ? ( ) - ( x )

Method: OECD TG 474 "Genetic Toxicology: Micronucleus Test".GLP: Yes ( ) No ( x ) ? ( )Test substance: As prescribed by 1.1-1.2 (from Rhône-Poulenc)

Purity: no data. (Most probably > 99.6%)Remarks: 10 mice per group.

Reference: Marzin, 1979b.

(b)Type: Micronucleus assaySpecies/strain: Mouse, BDF1Sex: Female ( ); Male ( x ); Male/Female ( ); No data ( )Route of administration: Gavage (orally)Exposure period: Post-treatment: Vanillin was administered every 3 hour (0, 3, 6, 9, 12,

15, 18 and 21 h) after MMC injection. Bone marrow cells were sampled24 hours after injection of MMC (mitomycin C).

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Time course study: Vanillin was given 7.5 and 9 hours after MMCinjection, and the bone marrow cells were sampled at 12, 16, 20, 24, 30,36, 48 and 72 hours after administration of MMC.

Doses: Post treatment: 125, 175, 250, 350 and 500 mg/kgTime course study: 500 mg/kg

Results: NegativePost treatment:Vanillin did not induce any micronucleated polychromatic erythrocytes(MN-PCEs).Post-treatment with Vanillin oral dose 500 mg/kg, 7.5 hours after i.p. injection of 2 mg/kg of MMC, caused about 50% decrease in the frequency of MN-PCEs.Time course study:The suppressing effect was not due to a delay in the formation of MN-PCEs by the cytotoxic action of Vanillin. Vanillin acts as an anticlastogenic factor in vivo.


Method: Micronucleus assay according to method described by Schmid (1976).GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo, Japan.

Purity: no dataRemarks: Mice: 7 weeks of age.

3 mice in each experimental group.Reference: Inouye et al, 1988.

(c)Type: Micronucleus assaySpecies/strain: Mouse ddYSex: Female ( ); Male ( x ); Male/Female ( ); No data ( )Route of administration: Gavage (orally)Exposure period: Post treatment: Vanillin was administered every 3 hour (0, 3, 6, 9, 12,

15 and 18 h) after irradiation. Bone marrow cells were sampled 24 hoursafter irradiation.Time-course study: Vanillin was given immediately after the irradiation,and bone marrow cells were sampled periodically (8, 12, 16, 24, 30, 36,48, 72 hours) after irradiation.

Doses: Post treatment: 250, 313 and 500 mg/kgTime-course study: 500 mg/kg

Results: Negative.Suppressing effects on X-ray-induced micronucleiPost treatment: The most pronounced reduction in the frequency ofMNPCEs was observed when Vanillin was administered to mice justafter irradiation (0 hours) (decrease: 55%).Significant suppression until 9 hours after irradiation.Testing at 0 hours:250 mg/kg: 42% decrease in MNPCEs.313 mg/kg: 42% decrease in MNPCEs.500 mg/kg: 55% decrease in MNPCEs.Time-course study: The observed reduction of MNPCEs was not not a reflection of the toxic effect of vanillin to the bone marrow.

Effect on mitotic

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index or P/N ratio:Genotoxic effects: + ( ) ? ( ) - ( x )

Method: Mouse micronucleus test according to Schmid (1976).GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo, Japan

Purity: no dataRemarks: Mice: 8 weeks of age.

Irradiated with X-rays at 200 rad.Reference: Sasaki et al, 1990b.

(d)Type: Mouse spot testSpecies/strain: Mouse, male PW, female C57BL/10 (mated)Sex: Female ( x ); Male ( ); Male/Female ( ); No data ( )Route of administration: Gavage (orally)Exposure period: I.p. injection with ENU (ethylnitrosourea) on the 10th day of pregnancy;

received 3 successive oral administrations of vanillin at 0, 4 and 24hours after ENU injection.

Doses: 0, 125, 250, 350 and 500 mg/kgResults: Negative.

Vanillin was shown to act as an antimutagen in vivo.Three successive oral administrations of Vanillin at 500 mg/kg, 0, 4 and24 hours after ENU injection decreased by approx. 50% the number ofmice with RS induced by ENU at 50 mg/kg.This suppression was observed at >= 250 mg/kg of Vanillin.Vanillin did not affect the number of mice with WMVS and pups perfemale, which indicates that Vanillin did not have any toxic effects onthe embryo.


Method: Antimutagenic effect in mouse spot test; in vivo method to detectsomatic cell mutations.The appearance of recessive color spots (RS) and white midventral spots(WMVS) was examined in pups aged about 30 days.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo, Japan

Purity: no dataRemarks: 8-16 week-old males mated with 8 week-old female.

Male (PW) has a recessive homozygous loci for coat colours and morphological characters.


(e)Type: Ring-X loss test in Drosophila melanogaster spermatozoaSpecies/strain: Drosophila melanogaster Female (C1), male (C1) - matedSex: Female ( x ); Male ( ); Male/Female ( ); No data ( )Route of administration: Oral feedExposure period: 48 hoursDoses: 0.1, 0.5, 1 and 2%Results: - Suppressive effect of Vanillin on ring-X loss that occurs spontaneously

in spermatozoa of D. melanogaster: Inhibition of 36%, 38%, 56% and59% at concentrations of 0.1%, 0.5%, 1% and 2%, respectively.

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- Significant suppressive effect of Vanillin on mitomycin C inducedring-X loss (when males treated with MMC were mated with thevanillin-treated females): Inhibition of 32% and 49% at concentrationsof 0.5% and 1%, respectively. However, this decrease was observedonly in the first 3 days after the interruption of the female's treatmentwith Vanillin. In the subsequent 3 days the frequencies of ring-X losswere not altered. On the other hand, vanillin at 0.1% did not show anyeffect on MMC-induced ring X-loss.- In contrast, Vanillin did not show any effect on chromosome loss provoked by Methyl methanesulphonate.


Method: No dataGLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Vetec Quimica Fina Ltd., Brazil

Purity: no dataRemarks: Males (aged 42-48 hours): fed with MMC (mitomycin C) and MMS

(methyl methanesulfonate) for 24 hours.Females (18.24 hours): fed with test solutions for 48 hours (new solutionafter 24 h).Mated.

Reference: De Andrade et al, 1992.

5.7 CARCINOGENICITY

(a)Species/strain: Mouse, S-strainSex: Female ( ); Male ( ); Male/Female ( ); No data ( x )Route of administration: DermalExposure period: 3 weeksFrequecy of treatment: Total 10 applicationsPostexposure observ. period: 18 weeksDoses: Roughly 3000 mg/kg bw per application; total dose of 600 mgControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( x ); Historical ( )Results: No co-carcinogenic effect.

No significant increase in local tumours.The incidence of tumours of the lung (the only organ examined in detail)was evidently unaffected by the treatment.

Method: Co-carcinogenic assay.Application of solutions, recording of tumours, examination of mice forlung adenomas at post-mortem, and histological examination.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from L. Light and Co. Ltd.

Purity: no dataRemarks: A skin painting study.

20 mice.Applications three times a week, total 10 applications.20 mice.A concentration of 20% in acetone, application of 0.3 ml (roughly 3000mg/kg bw/application, total Vanillin dose of 600 mg/mouse).

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Subsequent 18 weekly applications of 0.3 ml croton oil. First croton oil application 25 days after first application of Vanillin.

Reference: Salaman et al, 1956.

(b)Species/strain: Mouse, A/HeSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )Route of administration: i.p. injectionExposure period: 8 weeksFrequecy of treatment: 3 times a weekPostexposure observ. period: 16 weeksDoses: 150 and 750 mg/kg bw (Total doses (24 injections): 3600 and 18,000

mg/kg bw)Control group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( x ); Concurrent vehicle ( x ); Historical ( x )Results: Not carcinogenic.

Vanillin did not significantly induce lung tumours in mice.Method: Strain A mouse lung tumour bioassay.

Test for carcinogenicity by the pulmonary tumour response in strain A mice.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from JT Baker, lot no. 2-0022.

Purity: Ranging from 85-99%, majority 95-99% (several chemicals tested).

Remarks: Strain A mice have high spontaneous tumour incidence.Mice: 6-8 weeks old, average weight 18-20 g.15 male and 15 female per dose level.Vehicle: tricaprylin

Reference: Stoner et al, 1973, Stoner, 1991.

(c)Species/strain: Rat, Osborne-MendelSex: Female ( ); Male ( ); Male/Female ( x ); No data ( )Route of administration: Oral feedExposure period: 2 yearsFrequency of treatment: DailyPostexposure observ. period: NoneDose: 5000, 10,000 and 20,000 ppmControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( x ); Historical ( )

Results: No carcinogenic or toxic effects.No effect on growth or haematology.No macroscopic or microscopic changes in the tissues (incl. testes).

Method: 12 male and 12 female rats (test and control groups).Vanillin was mixed in the diet. Fresh diets were made and distributedweekly. The concentration was adjusted so that all rats received aconstant volume of 1 ml of solution/kg daily.3% (w/w) propylene glycol added to control or test diets as a binder to reduce evaporation of the flavour.The rat's weight, food intake and general condition were recorded everyweek.Haematological examinations were made at 3, 6, 12 and 22 months.

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These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.At the termination of the experiments the rats were sacrificed andexsaguinated. The tissues of all the rats were examined macroscopicallyat the time of sacrifice.The viscera were removed and the liver, kidneys, spleen, heart and testeswere weighed.These organs, the remaining abdominal and thoracic viscera, and onehind leg, for bone, bone marrow, and muscle, were preserved in 10%buffered formalin-saline solution for histopathological examination.For routine histopathology, sections were embedded in paraffin wax andstained with haematoxylin and eosin.Detailed microscopic examinations in the subacute studies weregenerally done on 6 or 8 rats, evenly devided by sex, from the high dosegroup and the control group.


(d)Species/strain: Rat, Osborne-MendelSex: Female ( ); Male ( x ); Male/Female ( ); No data ( )Route of administration: Oral feedExposure period: 1 yearFrequency of treatment: DailyPostexposure observ. period: NoneDose: 20,000 and 50,000 ppmControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( x ); Historical ( )Results: No carcinogenic or toxic effects.

No effect on growth or haematology.No macroscopic or microscopic changes in the tissues (incl. testes).

Method: 5 male rats (test and control groups).Vanillin was mixed in the diet. Fresh diets were made and distributedweekly. The concentration was adjusted so that all rats received aconstant volume of 1 ml of solution/kg daily.3% (w/w) corn oil added to control or test diets as a binder to reduce evaporation of the flavouring.The rat's weight, food intake and general condition were recorded everyweek.Haematological examinations were made at termination of the subacutestudies, and at 3, 6, 12 and 22 months in the chronic experiment.These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.At the termination of the experiments the rats were sacrificed andexsaguinated. The tissues of all the rats were examined macroscopicallyat the time of sacrifice.The viscera were removed and the liver, kidneys, spleen, heart and testeswere weighed.These organs, the remaining abdominal and thoracic viscera, and onehind leg, for bone, bone marrow, and muscle, were preserved in 10%buffered formalin-saline solution for histopathological examination.

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For routine histopathology, sections were embedded in paraffin wax andstained with haematoxylin and eosin.Detailed microscopic examinations in the subacute studies weregenerally done on 6 or 8 rats, evenly devided by sex, from the high dosegroup and the control group.


(e)Species/strain: Rat, Fischer 344Sex: Female ( ); Male ( x ); Male/Female ( ); No data ( )Route of administration: Oral feedExposure period: 4 weeksFrequecy of treatment: DailyPostexposure observ. period: NoneDoses: 2.0%Control group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( x ); Concurrent vehicle ( x ); Historical ( )Results: No significant increase in the thickness of the forestomach mucosa in the

prefundic or mid regions.In the glandular stomach, thickness and labeling indices weresignificantly increased compared to the diet without vanillin andNaNO2.

No significant increase in the thickness or labeling indices in the esophagus.

Method: Short term stomach cell proliferation study.The combined effects of Vanillin and NaNO2 on cell proliferation in the

upper digestive tract were examined. Groups of 5 rats were givenVanillin or basal diet either alone or in combination with 0.3% NaNO2for 4 weeks, and then killed.Mucosal thickness and proliferative indices in upper digestive tract (forestomach, glandular stomach and esophagus) were measured.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo, Japan.

Purity: > 97.0%Remarks: Rats: 5 weeks old.

Vehicle: NaNO2.

NaNo2 can stimulate phenolic compound-induced cell proliferation in

the upper digestive tract, particularly in the forestomach epithelium.Reference: Kawabe et al, 1994.

(f)Species/strain: Mouse, SKH/HR1Sex: Female ( x ); Male ( ); Male/Female ( ); No data ( )Route of administration: Oral feedExposure period: 40 weeksFrequecy of treatment: ConstantPostexposure observ. period: NoneDoses: 0.5% in dietControl group: Yes ( x ); No ( ); No data ( )

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Concurrent no treatment ( x ); Concurrent vehicle ( ); Historical ( )Results: No significant effect on weight.

Vanillin was not toxic to liver, due to no significant effect on liverweight (week 40).1) Vanillin did not reduce tumour latency (median tumour latency time:21.5 weeks, 20.3 in control) but significantly reduced tumourmultiplicity (week 21)(48%, p<0.05).2) Vanillin was without effect.

Method: Antiphotocarcinogenic and photoprotective properties.1) The animals received constant, daily (5 days a week) sub-erythemic levels of UVB radiation from Westinghouse BZS-WLG lamps.Radiation was discontinued when 25 sunburn units had been delivered atweek 11.Animals were evaluated weekly for tumor latency and multiplicity.Hepatomegaly was used as an indication to liver toxicity.2) Epidermal ornithine decarboxylase (ODC) assay:Groups of 3 mice with diets for 2 weeks, exposed to 0.45 J/cm2 of UVB,and episermal extract assayed for ODC activity 28 hours post-irradiation.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Aldrich Chemical Company, Milwaukee, WI, USA

Purity: no dataRemarks: Hairless mice, 18.5 to 20 weeks old.Reference: Black et al, 1986.

5.8 TOXICITY TO REPRODUCTION

Type: Fertility ( ); One generation study ( ); Two generation study ( );Other ( )

Species/strain:Sex: Female ( ); Male ( ); Male/Female ( ); No data ( )Route of administration:Exposure period:Frequecy of treatment:Postexposure observ. period:Premating exposure period: Male: Female:Duration of the test:Doses:Control group: Yes ( ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( ); Historical ( )NOEL Parental:NOEL F1 Offspring:NOEL F2 Offspring:Results: No data

General parental tox.:Toxicity to offspring:

Method:GLP: Yes ( ) No ( ) ? ( )Test substance:Remarks:Reference:

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5.9 DEVELOPMENTAL TOXICITY /TERATOGENICITY

(a)Species/strain: Mouse; male PW, female C57BL/10 (mated)Sex: Female ( x ); Male ( ); Male/Female ( ); No data ( )Route of administration: Gavage (orally)Duration of the test: From 10th day of pregnancy untill pups aged about 30 daysExposure period: I.p. injection with ENU (ethylnitrosourea) on the 10th day of pregnancy;

received 3 successive oral administrations at 0, 4 and 24 hours afterENU injection.

Frequecy of treatment: 3 administrations in 24 hoursDoses: 0, 125, 250, 350 and 500 mg/kgControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( x ); Concurrent vehicle ( ); Historical ( )One control group were treated with solvents for ENU and Vanillin, andone with solvents for ENU only.

NOEL Maternal Toxicity: > 1500 mg/kg bw/dayNOEL Teratogenicity: > 1500 mg/kg bw/ dayResults: Negative.

Vanillin was shown to act as an antimutagen in vivo.Three successive oral administrations of Vanillin at 500 mg/kg, 0, 4 and24 hours after ENU injection decreased by approx. 50% the number ofmice with RS induced by ENU at 50 mg/kg.This suppression was observed at >= 250 mg/kg of Vanillin.Vanillin did not affect the number of mice with WMVS and pups perfemale, which indicates that Vanillin did not have any toxic effects onthe embryo.

Maternal general tox.:Pregnancy/litter data:Foetal data:

Method: Antimutagenic effect in mouse spot test; in vivo method to detectsomatic cell mutations.The appearance of recessive color spots (RS) and white midventral spots(WMVS) was examined in pups aged about 30 days.

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo Japan

Purity: no dataRemarks: 8-16 week-old males mated with 8 week-old female.

Male (PW) has a recessive homozygous loci for coat colours and morphological characters.


(b)Species/strain: Mouse, ICRSex: Female ( x ); Male ( ); Male/Female ( ); No data ( )Route of administration: i.p.Duration of the test: 18 daysExposure period: Day 11 of gestationFrequecy of treatment: Single i.p. injectionDoses: 50 mg/kgControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( ); Concurrent vehicle ( x ); Historical ( )

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1) Saline only2) Saline + MNNG (40 mg/kg)3) Saline + MNNG (60 mg/kg)

NOEL Maternal Toxicity:NOEL Teratogenicity: >= 50 mg/kg bw/dayResults: 1) The effect of Vanillin on fetal development in mice - control with

saline only:Effects of Vanillin was comparable to that of saline control with respectto the number of live fetuses, fetal body weight, fetal mortality andincidence of external and skeletal abnormalities.

Maternal general tox.:Pregnancy/litter data: No. of litters: 3Foetal data: Mean no. of live fetuses: 13.7 +/- 1.8

Fetal body weight: 1.42 +/- 0,06 gFetal mortality: 9.6 +/- 4.8 %External abnormalities: 0 %Skeletal abnormalities: Malformations: 0 %

Variations: 25.7 +/- 17.4 %

2) The influence of Vanillin on MNNG-induced fetal defects (40 mg/kg MNNG):The incidence of external malformations in MNNG+Vanillin group wascomparable to that in MNNG+saline group.


Fetal body weight: 1.21 +/- 0.04 gFetal mortality: 10.2 +/- 2.6 %External abnormalities: 30.4 +/- 10.2 %

Brain: 0 %Face: 4.0 +/- 2.7 %Forelimb: 27.5 +/- 8.4 %Hindlimb: 27.7 +/- 10.1 %Others: 1.3 +/- 1.3 %

3) The influence of Vanillin on MNNG-induced fetal defects (60 mg/kg MNNG):The incidence of external malformations in MNNG+Vanillin group wascomparable to that in MNNG+saline group. The incidence ofmicrocephaly and facial defects, hshowed a tendency to decrease withVanillin.Fore- and hindlimbs:Significant decrease of syndactyly, decrease in oligodactyly noted, significant decrease of the incidence of cleft palate.Bractylyly was higher in the fore- and hindlimbs in MNNG (60 mg/kg)+Vanillin compared to MNNG (60 mg/kg)+saline group.


Fetal body weight: 1.06 +/- 0.06 gFetal mortality: 11.5 +/- 1.8 %External abnormalities: 67.5 +/- 11.2 %

Brain: 1.8 +/- 1.8 %

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Face: 25.6 +/- 6.1 %Forelimb: 56.2 +/- 10.2 %Hindlimb: 67.5 +/- 11.2 %Others: 26.4 +/- 9.9 %

Method: Examination of whether vanillin, which have mutation suppressingeffect, can modify the teratogenicity in mice caused by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).2) and 3) MNNG was administered on day 11 of gestation, Vanillin wasadministered one hour later.Killing of dams and examination of fetuses at day 18 of gestation.The number of implants, resorptions, dead fetuses and live fetuses werecounted.Live fetuses were weighed and examined for external malformationsunder a dissecting microscope, and then cleared and stained by means ofDawson's technique (Dawson, 1926) for skeletal examination.Furthermore, examination of the phalanges stained cartilagious and ossified components in the fore- and hindlimbs (modified method of Burdi, 1965).

GLP: Yes ( ) No ( ) ? ( x )Test substance: Vanillin from Tokyo Kasei Kogyo, Tokyo Japan

Purity: no data.Remarks: Females (9-13 weeks old) mated with males. Day 0 of gestation.Reference: Morita et al, 1988.

(c)Species/strain: Single-Comb White Leghorn chickens(chicken embryo)Sex: Female ( ); Male ( ); Male/Female ( ); No data ( x )Route of administration: Injection into yolk and through air cellDuration of the test: HatchingExposure period: Preincubation 0 hour and at 96 hoursFrequecy of treatment: Single injectionDoses: 5 dose levels up to 10 mg/eggControl group: Yes ( x ); No ( ); No data ( )

Concurrent no treatment ( x ); Concurrent vehicle ( x ); Historical ( )NOEL Maternal Toxicity: n.a.NOEL Teratogenicity: > 10 mg/eggResults: Vanillin showed no teratogenic response.

(Other: LD50 = 0.82 mg/egg by injection through air cell at 0 hour.)Maternal general tox.: n.a.Pregnancy/litter data:Foetal data:

Method: Study of teratogenicity of Vanillin in the developing chicken embryo.Calculation of the percentage mortality at each dose level.Calculation of the total number of birds having one or moreabnormalities.Calculation of the total number of birds having a structural abnormaltyof the head, viscera, limbs or body skeleton.

GLP: Yes ( ) No ( x ) ? ( )Test substance: Vanillin from Ruger Chemical Co, Irvington, NJ, USA

Purity: food grade quality, no further data.Remarks: Study of toxicity and teratogenicity of 80 food additive chemicals,

including Vanillin, in the developing chicken embryo.Four test conditions were used:

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- injection via the air cell- injection via the yolk- pre-incubation (0 hours)- 96 hours of incubation timeFor each condition at least 100 chicken embryos per dose level (at least5) were treated.

Reference: Verrett et al, 1980.

5.10 OTHER RELEVANT INFORMATION

A. Specific toxicities

(a)Type: ImmunotoxicityResults: No significant difference between group treated with Vanillin and

untreated control group.Remarks: Female CD-1 mice (30 mice/group).

Dosed intragastrically at 3 concentrations: 250, 500 and 1000 mg/kgVanillin/day and vehicle control (1% methylcellulose) for 5 consecutivedays.Control untreated group, positive control group: host resistance toListeria monocytogenes.Anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) assay: specific cellular activity, and total speen activity.

Reference: Gaworski et al, 1994.

(b)Type: ImmunotoxicityResults: Vanillin directly suppressed the in vitro anti-sheep red blood cell

(SRBC) antibody response at nontoxic doses.Remarks: Twenty chemicals were examined for immunomodulatory effects using

the Mishell-Dutton in vitro antibody-producing assay.Vanillin (200 ug/culture) was found to be one of five chemicals testedthat directly suppressed the in vitro anti-SRBC antibody response atnontoxic doses.All of the five positive chemicals were reported to interrupt an earlyphase of the immune response.They had no effect on the actual release of anti-SRBC antibody.

Reference: Kutz et al, 1980.

(c)Type: ImmunotoxicityResults: Vanillin had an immunostimulatory effect.Remarks:Reference: Archer et al. 1978.

(d)Type: ImmunotoxicityResults: 1) Vanillin was classified as a weak modulator macrophage function.Remarks: 1) Examination of the immunomodulatory effects of 25 chemicals using

alteration of peritoneal macrophage function as the study endpoint.

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Vanillin and 4 other test substances were found to decrease thepercentage of mouse peritoneal macrophage capable of ingesting yeast,although not below 50% of control.LC50 = 2050 ug/ml (the chemical concentration which reduced microphage viability to 50% after 20 hours exposure).4 indices: ingestion index, phatocytic capacity, killing index, adherenceindex).Vanillin was classified as a weak modulator, due to causing significantmodulation of an index at noncytotoxic doses, but the EC50 = 1/2 LC50,

and thus the modulatory index >= 0.50.Effective doses: 1, 10, 100, 300 and 1000 ug/ml.2) Screening for immunomodulators: effects of xenobiotics onmacrophage chemiluminiscence in vitro.Peak and total macrophage chemiluminiscence after 20 hours chemical exposure: EC50 >= 1000 ug/ml (EC50 = 50% effective concentration).Concentration range: 0.1 - 1000 ug/ml.Test substance: >= 98% pure, from ICN Pharmaceuticals, Plainview, NY, USA.

Reference: 1) Tam et al, 1984.2) Tam et al, 1990.

(e)Type: CytotoxicityResults:Remarks: Toxicity values (a 10 point scale from 0 to 9) in 4 in vitro short term

tests:- Inhibition of cell growth in sarcoma BP8 ascitic cells.- Inhibition of the oxidative metabolism in hamster brown fat cells.- Membrane damage of human diploid embryonic lung fibroblasts.- Inhibition of ciliary activity using chicken embryo trachea.Vanillin scores were 3, 2, 0, 0, respectively.

Reference: Curvall et al, 1984.

(f)Type: Other: Risk AssessmentResults:Remarks: In both Ames test and chromosome aberration tests, Vanillin showed no

mutagenic activity (Ishidate et al, 1984).Rats fed with 50,000 ppm Vanillin for 1 year or 10,000 ppm for 2 yearsshowed no deleterious effect (Hagan et al, 1967), pointing out Vanillinas a rather harmless food component or additive.

Reference: Feron et al, 1991.

(g)Type: Other: Antioxidant and pro-oxidant activityResults: Effects on free radicals, brain peroxidation and degradation of benzoate,

deoxyribose, aminoacids and DNA.Remarks: Test substance: from Wako Pure Chemicals Industries Ltd., Osaka

Japan.Reference: Liu et al, 1993.

(h)Type: Other: Effects on ciliary activity of the embryo chicken trachea in vitro.

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Results: Vanillin was found to be inactive on the ciliary activity within 60minutes.

Remarks: Testing principles:

The ciliary activity was studied at 37oC by means of invertedmicroscopy (magnification x 250).One tracheal ring from chicken tracheal organ cultures (16-17 days oldchicken embryos), was placed in a perpex testing chamber (Vol. 3.1 ml)containing the medium admixed with an ethanol or DMSO solution ofeach compound, at the same concentration; 5 mmol/l.The ciliary activity was displayed continuously on a TV-monitor duringthe entire exposure time, maximized to 60 minutes.Replicate tests were performed to at least 3 occasions, each experiment involving rings from different tracheal preparations.Both solvents were nontoxic to the cilia at the concentrations used in theexperiment.Test substance: all the compounds were checked for purity by TLC, GCand NMR.

Reference: Petterson et al, 1982.

B. Toxicodynamics, toxicokinetics

(a)Type: MetabolismResults:Remarks: Vanillin is metabolized by various mammalian species to a number of

urinary products, primarily vanillic acid in both free and conjugated forms. Other metabolites are conjugated vanillin, conjugated vanillylalcohol and catechol.I.p. injection of a single 100 mg/kg dose of vanillin in propyleneglycol/water solution to Sprague-Dawley rats resulted in the urinaryexcretion of free and conjugated vanillic acids (17 and 24% of dose),free and conjugated cathechol (trace and 4%) and conjugates of bothvanillyl alcohol (10%) and vanillin (6.5%) within a 24 hour period.

Reference: Wong et al, 1966.

(b)Type: MetabolismResults: Formaldehyde formation for Vanillin was only as a trace (= 70 to 200

pmol/mg microsomal protein/minute) with rat nasal microsomalenzymes, and 70 pmol/mg microsomal protein/minute with livermicrosomal enzymes.

Remarks: Screening assays to identify compounds which might be metabolized toformaldehyde in the nasal cavity.Test substance: from Sigma Chemical Co., St. Louis, MO USA.

Reference: Dahl et al, 1983.

(c)Type: MetabolismResults:Remarks: A single oral administration to male albino rats of 100 mg/kg bw

resulted in the urinary excretion of most of its metabolites within 24

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hours, mainly as glucuronide and/or sulphate conjugates, although theacids formed were also excreted free and as their glycine conjugates.In 48 hours, 94% of the dose had been excreted in the following proportions:- 47% Vanillic acid- 19% Vanillyl alcohol- 10% Vanilloylglycine- 8% Catechol- 7% Vanillin- 2% 4-methylcatechol- 0.6% 4-methylguaiacol- 0.5% guaiacol.No metabolites were found in the urine collected 48-96 hours afterdosing.

Reference: Strand et al, 1975.

(d)Type: MetabolismResults:Remarks: The conversion of Vanillin to Vanillic acid was demonstrated in human

and rat liver cells in culture.Reference: BIBRA Toxicity Profile, 1990.

(e)Type: MetabolismResults:Remarks: An early study found only trace amounts of Vanillin in the urine of

rabbits given 2 g/day orally (about 1 g/kg bw/day), the Vanillin being conjugated with sulphuric acid.Most of the dose was oxidized to Vanillic acid, and this too was excretedas the ethereal sulphate conjugate.

Reference: BIBRA Toxicity Profile, 1990.

(f)Type: MetabolismResults:Remarks: Eighty-three percent of an oral dose (1g/kg) of Vanillin administered to

rabbits was accounted for after 24 hours in the urine.Vanillic acid was observed free (64%) and conjugated (25%) either as aglucuronide or a sulfate.Unchanged Vanillin (14%) was present in the urine, mostly as glucurovanillin, up to 6 hours after dosing, but its presence ceased abruptly at that time.

Reference: Sammons et al, 1941.

(g)Type: MetabolismResults:Remarks: The vanillic acid content in the 24 hour urine of one human maintained

on a plant free diet for 2 days dropped from ca 9 mg to 0.3 mg. At thistime an oral dose of 100 mg vanillin resulted in the urinary excretion of96 mg vanillic acid (ca 94% of the administered dose) in the next 24hour period. No unchanged vanillin was observed in the urine.

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Reference: Dirscherl et al, 1964.

(h)Type: MetabolismResults:Remarks: In vitro incubation of 0.3 mg vanillin with rat (Sprague-Dawley; M)

liver homogenates for 2 hours in a phosphate buffer at 37oC, followedby addition of 0.2 ml of concentrated HCl resulted in the formation ofvanillic acid with an 81% yield.

Reference: Dirscherl et al, 1966.

(i)Type: MetabolismResults:Remarks: Anaerobic incubation of 5-10 mg vanillin with .... caecal extracts for 46

hours resulted in the formation of vanillic acid, 4-methylguaiacol, 4-methylcatechol, protocatechuic acid, and a 4th unidentified product. Inaddition, catechol and unchanged parent compound were detected in 1 of3 experiments.

Reference: Scheline, 1972.

(j)Type: MetabolismResults:Remarks: Guinea pigs (390 g) were administered daily doses (oral implied) of 15

mg vanillin (385 mg/kg) for 10 days. A total of 200 mg pure vanillicacid and very slight amounts of benzoic acid were isolated from theurine.

Reference: Bernhard et al, 1955.

(k)Type: MetabolismResults:Remarks: Two human subjects, male and female, were placed on a plant free diet

72 hours prior to treatment and maintained on the diet for 24 hoursfollowing administration of vanillin. Treatment consisted of one subjectorally ingesting 60 ml of vanilla extract within 5 minutes, and secondingesting 10 average servings of an artificially flavoured vanilla puddingwithin 12 hours. Urine analysis of samples collected for a 24 hourperiod during and following both treatments revealed trace levels of 3-methoxy-4-hydroxy-benzylamine (vanillylamine) as a result of bothvanilla extract and pudding ingestion, respectively.

Reference: Perry et al, 1965.

5.11 EXPERIENCE WITH HUMAN EXPOSURE

(a)Results: No primary irritationRemarks: In closed-patch tests on human skin, vanillin caused no primary

irritation when tested at concentrations of 20% on 29 normal subjects, of2% on 30 normal subjects and of 0,4% in 35 subjects with dermatoses.

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No further details known.Reference: Opdyke, 1977.

(b)Results: No sensitizationRemarks: Maximation tests were carried out on groups of 25 volunteers.

The material was tested at concentrations of 2% and 5% in petrolatum and produced no sensitization reactions.

Reference: Opdyke, 1977.

(c)Results: No sensitization or irritationRemarks: Vanillin applied undiluted for 48 hours in the standard occluded

aluminium patch test used by the North American Contact Dermatitis Research Group (NACDRG) did not produce any irritation or sensitization in a 62 year old subject with a perfume dermatitis.


(d)Results:Remarks: Positive reactions to Vanillin were reported in eight out of 142 patients

who were already senstized to Balsam of Peru.In studies of sensitization to Balsam of Peru and its components,Vanillin (pure or 10% in vaseline) produced positive patch test reactionsin 21 out of 164 patients sensitive to the balsam. Vanillin wasconcidered to be a secondary allergen, since sensitivity was found onlyin patients sensitive to Vanilla, isoeugenol and coniferyl benzoate.Cross-sensitization to other substituted benzaldehydes was particularlyuncommon. Vanillin was found not to be responsible for most cases ofsensitivity to natural Vanilla.


(e)Results:Remarks: In well-conducted (double-blind) challenge tests, an asthmatic patient

was given Vanillin at 1.5 hours intervals, two or three times, providingno reaction occured within 15 minutes of a challenge. There was some evidence that Vanillin reduced lung function at oral doses of 0.24 and 1mg. Itching of the ears and throut was also described.

Reference: BIBRA Toxicity Profile, 1990.

(f)Results:Remarks: Chinese manufacturer of Vanillin - packaging section:

Persons tested:- Persons who have/had dermatitis (working in the packaging section).- Control group: Healthy workers from other units.Test material removed 48 hours after application.Results read 1 hour later.Negative in both groups.From the negative results of the above patch tests with Vanillin, it isconcidered that the dermatitis occuring in the packaging section may be

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due to mechanical irritation of Vanillin dust stuck on the skin, ratherthan chemical irritation or sensitization.The itching subsiding after taking shower in most of the workers supportthe above postulation.


(g)Results:Remarks: Bronchospasm was reported caused by Vanillin mixed with lactose in a

controlled double-blind challenge test in a 52 year old asthmatic patient.However, the patient also reacted to the "placebo", lactose.

Reference: Van Assendelft, 1984.

6. REFERENCES

ACF ChemieFarma NV, Netherlands. Product data sheet. Vanillin ex lignin. 0882/6.

Ash, M&I. 1995. Handbook of Food Additives. 783.

Archer, D.L. et al. 1978. Use of an in vitro Antibody-Producing System for Recognizing PotentiallyImmunosuppressive Compounds. Int. Archs. Allergy Appl. Immun. 56: 90-93.

Babich, H. et al. 1993. Comparative cytotoxicities of selected minor dietary non-nutrients withchemopreventive properties. Cancer Lett. (Shannon Irel.). 73 (2): 127-133.

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Weast, R.C. et al. 1989. Handbook of Data on Organic Compounds. 2nd ed. CRC Press. (Citation:Euclid Data Sheet for Vanillin by Rhône-Poulenc Chimie, created 26.01.95, updated 01.06.95)

Wong, K.P. et al. 1966. Metabolism of Vanillin and related substances in the rat. Can. J. Biochem. 44(5): 635-644. (Citation: FEMA. 1985. Scientific Literature Review of Vanillin and Derivatives)

Younger Laboratories Inc. 1976. Report no. Y-76-261. Unpublished report (Monsanto ChemicalCompany, USA). (Citation: Euclid Data Sheet for Vanillin by Rhône-Poulenc Chimie, created 26.01.95,updated 01.06.95)

OECD SIDS VANILLIN

bli i116

EXTRACT FROM IRPTC LEGAL FILE

OECD SIDS VANILLIN

bli i117

File: 17.01 LEGAL rn : 401061

systematic name:Benzaldehyde,4-hydroxy-3-methoxy- common name :vanillin reported name :VANILLIN cas no :121-33-5 rtecs no :YW5775000 area : CZE type : REG -------------------------------- |subject|specification|descriptor| |-------+-------------+----------| | FOOD | | MPC | -------------------------------- LIMIT OF ADDITIVE PRESENT DUE TO PRODUCTION, PACKING, TRANSPORT AND STORAGE OF FOOD PRODUCTS: 1.0G/KG. entry date: DEC 1991 effective date: 1JUL1986

title: DIRECTIVE NO. 50/1978 ON FOREIGN SUBSTANCES IN FOODSTUFFS original : HPMZC*, HYGIENICKE PREDPISY MINISTERSTVA ZDRAVOTNICTVI CSR(HYGIENIC REGULATIONS OF MINISTRY OF HEALTH OF CSR), 43 , , , 1978 amendment: HPMZC*, HYGIENICKE PREDPISY MINISTERSTVA ZDRAVOTNICTVI CSR(HYGIENIC REGULATIONS OF MINISTRY OF HEALTH OF CSR), 61 , , , 1986

*******

File: 17.01 LEGAL rn : 1122371

systematic name:Benzaldehyde,4-hydroxy-3-methoxy- common name :vanillin reported name :VANILLIN cas no :121-33-5 rtecs no :YW5775000 area : RUS type : REG -------------------------------- |subject|specification|descriptor| |-------+-------------+----------| | AIR | OCC | MAC | | | | CLASS | -------------------------------- CLV : 1.5 MG/M3 (VAPOUR, AEROSOL) HAZARD CLASS: III entry date: MAY 1990 effective date: 01JAN1989

amendment: GOSTS*, GOSUDARSTVENNYI STANDART SSSR(STATE STANDARD OF USSR), 12.1.005 , , , 1988

*******

File: 17.01 LEGAL rn : 1504568

systematic name:Benzaldehyde,4-hydroxy-3-methoxy- common name :vanillin reported name :VANILLIN cas no :121-33-5 rtecs no :YW5775000 area : F/W type : REC -------------------------------- |subject|specification|descriptor| |-------+-------------+----------| | FOOD | ADDIT | ADI | | FOOD | ADDIT | MXL | | USE | ADDIT | PRMT | --------------------------------

OECD SIDS VANILLIN

bli i118

USE: FLAVOUR; ADI: 0-10 MG/KG BW; MXL: JAMS AND JELLIES, CREAM: LIMITED BY GOOD MANUFACTURING PRACTICE; CHOCOLATE, COCOA POWDERS AND DRY COCOA-SUGAR MIXTURES, COMPOSITE AND FILLED CHOCOLATE, COCOA MASS AND COCOA PRESS CAKE: IN SMALL AMOUNTS TO BALANCE THE FLAVOUR; CANNED BABY FOODS, PROCESSED CEREAL-BASED FOODS FOR INFANTS AND CHILDREN: 70 MG/KG OF READY-TO-EAT PRODUCT entry date: MAY 1991 effective date: 1983

amendment: FAOCA*, CODEX ALIMENTARIUS, XIV , , 262 , 1983

Documents

VANILIN CAS N°: 121-33-5 - inchem.orginchem.org/documents/sids/sids/121335.pdf · oecd sids vanillin bli i 2 sids initial assessment profile cas no. 121-33-5 chemical name vanillin